Bipartite molecules and uses thereof in treating diseases associated with abnormal protein aggregates

ABSTRACT

Bipartite molecules comprising a peptide affinity moiety and at least one charged moiety and uses thereof in reducing formation of abnormal protein aggregate and treating diseases associated with such abnormal protein aggregate, including neurodegenerative disease characterized by formation of protein aggregates.

CROSS REFERENCE TO RELATED APPLICATION

This PCT application claims the benefit of U.S. Provisional ApplicationNo. 62/029,030, filed Jul. 25, 2014, under 35 U. S. C. §119. The entirecontent of the prior application is herein incorporated by reference.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases, such as Alzheimer's disease (AD),Huntington's disease (HD), synucleinopathy (e.g., Parkinson's disease(PD) and dementia with Lewy bodies (DLB)), tauopathy (e.g., Pick'sdisease, progressive supranuclear palsy, corticobasal degeneration, andfrontotemporal dementia with Parkinsonism linked to chromosome 17),TDP-43 proteinopathy (e.g., amyotrophic lateral sclerosis (ALS) andfrontotemporal lobar degeneration with ubiquitinated inclusions(FTLD-U)), and Creutzfeldt-Jacob disease (CJD), are characterized by anabnormal aggregation of pathogenic proteins, leading to the formation ofinclusion bodies (IBs). Recently, the prion-like behavior of abnormalprotein aggregates has been established, showing that these IBs not onlyserve as a diagnostic pathological marker, but also play an importantrole in the pathogenesis of these diseases.

Because neurodegenerative diseases are usually age-related, theyprimarily affect patients in mid- to late-life. It is expected thattheir incidence will increase as the population ages. Since theprocesses of many neurodegenerative diseases are not well-understood,there is currently no cure for these diseases. It is therefore of greatimportance to develop effective therapies for neurodegenerativediseases.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the unexpecteddiscovery that a number of bipartite molecules, e.g.,polyR-Aβ40-(25-35), PEI-V24P (10-40), and polyR-polyQ, successfullydecreased abnormal protein aggregation and demonstrated beneficialtherapeutic effects in murine models of Huntington's disease andAlzheimer's disease (APP/PS1). More specifically, polyR-polyQ was foundto bind mutant huntingtin (mHtt) protein aggregates, decreasemHtt-mediated toxicity, and delay the onset and progress of neurologicdysfunctions observed in the R6/2 murine model of Huntington's disease.Furthermore, polyR-Aβ40 (25-35) and PEI-V24P (10-40) were found toameliorate Aβ₄₀ cytotoxicity in mouse neuroblastoma cells, preventmemory deterioration, and decrease the level of Aβ plaque in the brainsof the APP/PS1 transgenic murine model of Alzheimer's disease. Thebipartite molecules described herein all contain an affinity moiety(e.g., the polyQ portion, the Aβ40 (25-35) portion, and the V24P (10-40)portion) capable of binding to an abnormal protein aggregate or acomponent thereof (e.g., a monomer of the aggregate) and a chargedmoiety (e.g., the polyR portion or the PEI portion).

Accordingly, one aspect of the present disclosure relates to a bipartitemolecule comprising (i) a peptide affinity moiety that binds to adisease-associated abnormal protein aggregate, or component thereof; and(ii) at least one charged moiety. The affinity moiety is linked (e.g.,covalently) to the at least one charged moiety. In some examples, thebipartite molecule described herein may contain one charged moiety(e.g., a charged peptide fragment), which may be conjugated to eitherthe N-terminus or the C-terminus of the peptide affinity moiety. Inother examples, the bipartite molecule described herein may contain twocharged moieties (e.g., the same or different), one being conjugated tothe N-terminus of the peptide affinity moiety and the other beingconjugated to the C-terminus of the peptide affinity moiety.

In some embodiments, the peptide affinity moiety binds an abnormalprotein aggregate or a component thereof that is associated with aneurodegenerative disease (e.g., Alzheimer's disease, Huntington'sdisease, synucleinopathy (e.g., Parkinson's disease, and dementia withLewy bodies), tauopathy (e.g., Pick's disease, progressive supranuclearpalsy, corticobasal degeneration, and frontotemporal dementia withParkinsonism linked to chromosome 17), TDP-43 proteinopathy (e.g.,amyotrophic lateral sclerosis (ALS), frontotemporal lobardegeneration-TDP-43 proteinopathy, frontotemporal lobardegeneration-tauopathy, Pick's disease, cortical basal degeneration,progressive supranuclear palsy, FTDP-17 with ubiquitinated inclusions(FTLD-U)), or Creutzfeldt-Jacob disease.

In some examples, the peptide moiety of the bipartite molecule is afragment of amyloid β or TDP-43, which can interfere with amyloid β orTDP protein aggregation. The fragment of amyloid β may comprise theamino acid sequence of GSNKGAIIGLM (SEQ ID NO: 1) orYEVHHQKLVFFAED^(D)PGSNKGAIIGLMVGGVV (SEQ ID NO: 2) (^(D)P refers to theD-form of proline), which is capable of interfering with amyloid βprotein aggregation.

In other examples, the peptide affinity moiety of the bipartite moleculedescribed herein can be a polyglutamine (PolyQ) fragment (containing,e.g., 5-20 Q residues such as 10 Q or 15 Q residues), which is capableof binding to the polyQ stretch of huntingtin protein, therebypreventing the formation of abnormal protein aggregates.

In any of the bipartite molecules described herein, the at least onecharged moiety of the bipartite molecule can be a polyarginine (PolyR)fragment (containing, e.g., at least 5, 8, 10, or 12 R residues) orpolyethylenimine (PEI). In some embodiments, the bipartite molecules maycontain more than 2 charged moieties (e.g., 2, 3, or more). Examples ofthe bipartite molecules as described herein include, but are not limitedto:

(SEQ ID NO: 3) RRRRRRRRGSNKGAIIGLM, (SEQ ID NO: 5)YEVHHQKLVFFAED^(D)PGSNKGAIIGLMVGGVV-PEI, (SEQ ID NO: 6)RRRRRRRRWDQQQQQQQQQQ, (SEQ ID NO: 7) RRRRRRRRWDQQQQQQQQQQQQQQQ,  or(SEQ ID NO: 4) RRRRRRRRGSNKGAIIGLM-PEI.

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising one or more bipartite molecules as describedherein and a pharmaceutically acceptable carrier.

In yet another aspect, the present disclosure provides a method forreducing the formation of an abnormal protein aggregate associated witha disease (e.g., a neurodegenerative disease) or treating such adisease, the method comprising administering (e.g., via an intranasalroute) to a subject in need of the treatment an effective amount of oneor more bipartite molecules as described herein. In some examples, thesubject is a human patient having, suspected of having, or at risk for,a neurodegenerative disease, e.g., Alzheimer's disease, Huntington'sdisease, Parkinson's disease, dementia with Lewy bodies, amyotrophiclateral sclerosis, frontotemporal lobar degeneration-TDP-43proteinopathy, frontotemporal lobar degeneration-tauopathy, Pick'sdisease, cortical basal degeneration, progressive supranuclear palsy,FTDP-17, and/or Creutzfeldt-Jacob disease.

In some examples, the neurodegenerative disease is Alzheimer's disease(AD) and the bipartite molecule for use in treating AD comprises anaffinity moiety having the amino acid sequence GSNKGAIIGLM (SEQ IDNO: 1) or YEVHHQKLVFFAED^(D)PGSNKGAIIGLMVGGVV (SEQ ID NO: 2). Such abipartite molecule can be RRRRRRRRGSNKGAIIGLM (SEQ ID NO: 3),RRRRRRRRGSNKGAIIGLM-PEI (SEQ ID NO: 4), orYEVHHQKLVFFAED^(D)PGSNKGAIIGLMVGGVV-PEI (SEQ ID NO: 5).

In another example, the neurodegenerative disease is Huntington'sdisease and the bipartite molecule for use in treating this diseasecomprise an affinity moiety having a PolyQ fragment. Such a bipartitemolecule can be RRRRRRRRWDQQQQQQQQQQ (SEQ ID NO: 6), orRRRRRRRRWDQQQQQQQQQQQQQQQ (SEQ ID NO: 7).

Also within the scope of the present disclosure are (a) pharmaceuticalcompositions for use in interfering with abnormal protein aggregationassociated with a disease (e.g., preventing the formation of, ordisrupting existing, aggregates) or treating such a disease, thepharmaceutical composition comprising a pharmaceutically acceptablecarrier and one or more bipartite molecule as described herein; and (b)uses of any of the pharmaceutical compositions or bipartite moleculesfor manufacturing a medicament for treating a disease associated withabnormal protein aggregation. Such a disease can be a neurodegenerativediseases characterized by abnormal protein, including, but not limitedto, Alzheimer's disease, Huntington's disease, Parkinson's disease,dementia with Lewy bodies, amyotrophic lateral sclerosis, frontotemporallobar degeneration-TDP-43 proteinopathy, frontotemporal lobardegeneration-tauopathy, Pick's disease, cortical basal degeneration,progressive supranuclear palsy, FTDP-17, and/or Creutzfeldt-Jacobdisease.

The details of one or more embodiments of the disclosure are set forthin the description below. Other features or advantages of the presentdisclosure will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic illustration of the modular design of the therapeuticpeptide for HD.

FIG. 2. Stability of the therapeutic peptides determined by HPLC. A:diagrams showing the retention time of the tested peptides from day 0 today 28. B: charts showing the amount of the tested peptides at day 0 andday 28. Note the 8R5Q and 8R10Q remained stable in water for 28 days,but the soluble 8R15Q and 8R20Q gradually decreased with time.

FIG. 3. Diagrams showing the co-localization of tested bipartitepeptides with the 109QmHtt aggregates. Panel A: Structure ofTAMRA-labeled 8R10Q used in this study. Panel B: Epifluorescencemicrographs of Neuro2a cells expressing 109QmHtt treated withTAMRA-8R10Q (8R10Q) at various time points. Note the co-localization of8R10Q and 109QmHttGFP aggregates (GFP) from 8-24 hours (arrows). Scalebar: 10 μm. Panel C: TIRF micrographs of the Neuro2a expressing 109QmHtttreated with water or peptides as indicated at various time points. Notethe decrease in aggregate size in 8R10Q and 8R15Q-treated cells. PanelD: Quantitation of the size of individual aggregates in Neuro2a at 8hours (left) and 24 hours (right) after peptide treatment. The size wassignificantly decreased by 8R10Q or 8R15Q peptide at both time points.Panel E: Quantitation of the number of aggregates in 20 Neuro2a cells at12 hours (left) and 24 hours (right) after peptide treatment. Both the8R10Q and 8R15Q increased the number of aggregates. Statistics performedwith one way ANOVA. **p<0.01; ***p<0.001.

FIG. 4. Testing the ability of therapeutic peptides to decrease mHttaggregation in Neuro2a cells overexpressing mHtt. Panel A: Filter trapassays and corresponding quantitation of cells (panel B) treated asindicated at 8 hours (T1), 24 hours (T2), or both (T1+2) aftertransfection with the 109QmHtt construct. Both 8R10Q and 8R15Qsignificantly decreased mHtt aggregates when treated at T1 or both timepoints, but not at T2 alone. Panel C: Western blots and quantitation(panel D) of RIPA-soluble (sol) and—insoluble (ins) fractions of Neuro2acells overexpressing 25QHtt or 109QmHtt and treated as indicated. Thelevels of 109QmHtt in both the soluble and insoluble fractions weresignificantly decreased by 8R10Q. Panel E: Western blot of the 109QmHttin cells treated as indicated. Note that MG132 blocked the effect of8R10Q peptide with respect to decreasing insoluble 109QmHtt. Panel F:Quantitation of ratio of 109QmHtt in cells treated MG132 (+) over DMSO(−) in relationship to peptides. Statistical analysis conducted with oneway ANOVA, *<0.05, **<0.01, ***<0.001, ns: not significant.

FIG. 5. Therapeutic effect of 8R10Q and 8R15Q in 109QmHtt-expressingcells. Panel A: Cell viability assay by MTT in Neuro2a cells expressing25QHtt (left) or 109QmHtt (right) treated with H₂O₂ at 50 μM and theindicated peptide. Panel B: Cell growth curve of Neuro2a cellsexpressing 25QHtt or 109QmHtt treated with indicated peptide. The cellsin right subpanel were further treated with H₂O₂ at 12.51. μM 16 hoursafter transfection. 109QmHtt cells growth was slower than that of the25QHtt cells. 8R10Q treatment rescued the slow growth of Neuro2a cellsby 109QmHtt with or without H₂O₂. Panel C: Micrographs of the phasecontrast and fluorescent images of retinoic acid-differentiated Neu2acells expressing GFP-25QHtt or GFP-109QmHtt (GFP) treated with theindicated peptide. Note the neurites of the differentiated cells. PanelD: Quantitation of the percentage of differentiated cells with neurites.Fewer 109QmHtt-expressing cells had neurites vs. 25QHtt-expressingcells. 8R10Q treatment could significantly rescue the defect in neuriteoutgrowth. The experiments were conducted in triplicate and repeatedtwice. Statistical analysis was performed with one way ANOVA, *p<0.05;**p<0.01; ***p<0.001, ns: not significant.

FIG. 6. 8R10Q ameliorated functional deterioration of R6/2 transgenicmice. Panel A: Longitudinal rotarod performance of wild type (WT) andR6/2 mice treated with PBS or 8R10Q peptide. Note the significant delayin motor deterioration of R6/2 mice treated with 8R10Q from 11 weeks ofage. Panel B: T maze test of WT and R6/2 mice at 13 weeks of age. The8R10Q significantly rescued the memory deficit in R6/2 mice. Panel C:The curves of blood sugar in serum of WT and R6/2 mice treated asindicated. Note 8R10Q treatment significantly decreased the rise inblood sugar in R6/2 mice. Panel D: Lifespan of WT and R6/2 mice treatedwith PBS vs. 8R10Q. 100% of R6/2 mice died before 16 weeks of age. 8R10Qsignificantly extended the lifespan of R6/2 mice. WT mice N=6/group;R6/2 mice N=10/group; statistical analysis performed with two way ANOVA(panels A and C), one way ANOVA (panel B), *p<0.05; **p<0.01;***p<0.001, ns: not significant, and log-rank (Mantel-Cox) Test (D),***p<0.0001.

FIG. 7. Amelioration of neuronal damage of 13 week-old R6/2 mice by8R10Q peptide. Panel A: Micrographs of the Nissl stain sections ofcortex (CTX) of WT and R6/2 mice. The cortex and its thickness arehighlighted by the dashed lines and red solid lines, respectively. PanelB: Quantitation of the cortical thickness. Note the decrease in corticalthickness in R6/2 mice which was reversed by the 8R10Q. Panels C and E:Micrographs (panel D) and (panel F) show the corresponding quantitationof the Nissl stained sections of the cortex and striatum, respectively.Note the decrease in the number of neurons, which was reversed by 8R10Q.N=3/group, each bar represents the average of 5 sections. Statisticalanalysis performed with one way ANOVA, *p<0.05; ***p<0.001, ns: notsignificant.

FIG. 8. Decrease in mHtt aggregates and glial pathology in 13-week-oldR6/2 mice by 8R10Q peptide treatment. Panel A: Micrographs andcorresponding quantitation (panel B) of the immunostained sections ofcortex (CTX) and striatum with EM48 antibody. The brown dots show themHtt aggregates. The left subpanels of panel B show the quantitativedata with respect to the total area occupied by the aggregates, and theright, the intensity of individual aggregates. Panel C: Photographs ofthe immunofluorescent-stained sections of the cortex of R6/2 mice withanti-GFAP antibody. Panel D: Quantitation of the GFAP intensity showed asignificant decrease in 8R10Q treated mice. Panel E: Micrographs of theimmunostained sections of the cortex (CTX) and striatum of WT and R6/2mice with anti-Ibal antibody. Panel F: Quantitation of lbal intensityrevealed an obvious increase in lbal immunoreactivity in R6/2 mice whichwas reversed by the 8R10Q. N=3/group. Each bar represented an average of15 sections/mouse group. Statistics were performed with Student's t testfor panels B and D, but with one way ANOVA, *p<0.05; **p<0.01;***p<0.001, ns: not significant.

FIG. 9. Changes in the levels of Htt mRNA by quantitative RT-PCR inNeuro2a transfected with 25QHtt or 109QmHtt and treated with theindicated peptide. Statistical analysis was performed with one wayANOVA, ***p<0.001, ns: not significant

FIG. 10. Effect of the designed bipartite peptides on inhibition offibrillization. The peptides were dissolved in 20 mM sodium phosphatebuffer with 150 mM KCl (pH 7) and incubated at 25° C. CD spectra and TEMimages were taken for Aβ40 (panels A and D), R₈Aβ(25-35) (panels B andE), ^(D)R₈-Aβ (25-35) (panels C and F), and the 1:1 mixture of Aβ40 withR₈-Aβ (25-35) (panels G and J) or ^(D)R₈-Aβ (25-35) (panels H and K).The CD spectra were recorded at the indicated incubation time. The TEMimages were taken after prolonged incubation. Panel I: The time courseof amyloidogenesis of Aβ40 with and without the designed bipartitepeptides.

FIG. 11. Cell viability measurement by MTT assays. Panel A: Neuro2acells treated with DMSO (control), Aβ40, R₈-Aβ(25-35) or^(D)R₈-Aβ(25-35). Panel B: Neuro2a cells treated with DMSO (control),Aβ40, and Aβ40 with equal molar R₈-Aβ(25-35), ^(D)R₈-Aβ(25-35), orAβ(25-35). Each bar was generated by a triplicate experiment; the studywas repeated 3 times. The statistics were performed with one way ANOVAcorrected with Fisher's LSD test. ***:p<0.001; ****p<0.0001; ns, notsignificant.

FIG. 12. R₈-Aβ(25-35)-PEI improved memory as measured by the Morriswater maze assay. Panel A: Plot of the escape latency period of wildtype (WT) and APP×PS1 transgenic (Tg) mice of 8 months of age treatedwith either PEI or R₈-Aβ(25-35) peptide from the age of 4 months to 8months. Note a significant shortening of the latency in Tg mice by thetherapeutic peptide. Panel B: Percentage of time of WT or Tg micetreated as indicated spent in swimming in the target quadrant where thehidden platform used to be. The times of the indicated mice crossing thetarget quadrant. R₈-Aβ(25-35)-PEI peptide increased the time of Tg micein the target quadrant. Panel C: The percentage of time of the WT or Tgmice as indicated crossing the target quadrant. Ten mice per group wereused in this study. The statistics were performed with two way ANOVAwith Fisher's LSD posthoc analysis. N=10/group *<0.05; ***<0.0005;****<0.0001

FIG. 13. ELISA assay for the level of Aβ40 and Aβ42 in the cortex (panelA) and hippocampus (panel B) in the 8 month-old Tg mice treated with PEIor R₈-Aβ(25-35)-PEI. The therapeutic peptide significantly reduced thelevel of both Aβ40 and Aβ42 in both regions. N=3/group; *:p<0.05;**:p<0.01; ***:p<0.001; ****:p<0.0001; ns: not significant. Statisticsby Student's t-test.

FIG. 14. Effect of intranasally delivered R₈-Aβ(25-35)-PEI on APP×PS1mice on Aβ clearance after 4 months treatment. Wild type (WT) andAPP×PS1 transgenic (Tg) mice were treated with either PEI orR₈-Aβ(25-35)-PEI from the age of 4 months to 8 months. Panels A-E showThS-staining of the brain slices.

FIG. 15. Assays for the level of level of IL-6 and IL-1β in the cortex(N=3/group) in the 8 month-old Tg mice treated with PEI orR₈-Aβ25-35-PEI peptide from the age of 4 months to 8 months. As shown,the therapeutic peptide signficantly decreased the level of interleukinIL-6 and IL-1β in the cortex. N=3/group; **:P<0.01. Statistics byStudent's t-test.

FIG. 16. MicroPET amyloid images of 12 month-old wild type (WT) controlmice and transgenic (Tg) mice treated with PEI or R₈-Aβ(25-35)-PEIpeptide for 8 months. Panel A: Representative PET images of the Tg mousebrains co-registered with a mouse T2-weighted MRI brain template. Asshown, the brain of the PEI-treated Tg mouse had much higher amyloidsignals at the cortex (CT), hippocampus (HP), and amygdala (AMY)compared with that of the WT mouse. R₈-Aβ(25-35)-PEI peptide treatmentreduced the amyloid signal of the Tg mouse. Panel B: Quantitation of thesignal of ¹¹C-labled Pittsburg compound B (PIB) in regions as indicated.N=6 per group, **<0.001; ***<0.0005, statistics were conducted with theStudent's t test.

FIG. 17. ELISA assay of the level of total and insoluble Aβ40 and Aβ42in the cortex and hippocampus in the 12 month-old Tg mice treated withPEI or R₈-Aβ(25-35)-PEI for 8 months. ELISA assay of total Aβ₄₀ and Aβ₄₂in the cortex (panel A) and hippocampus (panel B) and insoluble Aβ₄₀ andAβ₄₂ in the cortex (panel C) and hippocampus (panel D). (N=5 per group).Statistics were conducted with the Student's t test. **:p<0.01;***:p<0.001; ns, not significant.

FIG. 18. Structural studies on V24P(1-28) and V24P(10-40). The peptidesat concentrations of 30 or 60 μM were incubated at 25° C. for theindicated times (0-12 days), then the CD spectra (panels A and C) andfluorescence spectra after binding ThT (panels B and D) were recorded.Panel E: Electron microscopy images of 60 μM V24P(1-28) and V24P(10-40)after incubation at 25° C. for about 1 month.

FIG. 19. Structural studies on V24P(13-36), V24P(16-33), andV24P(19-30). The peptides were dissolved at concentrations of 30, 60, or90 μM and their CD spectra (panel A) and fluorescence spectra afterbinding ThT (panel B) were immediately recorded.

FIG. 20. Cell viability assay. The viability of mouse N2a cellsincubated with the indicated peptide(s) was measured using the MTTassay. Panel A: Comparison of the viability of Aβ40 and the designedpeptides containing the V24→^(D)P mutation. Panel B: Comparison of theviability of 30 μM Aβ40 alone or together with 30 μM designed peptide.The standard deviations are shown as bars (compared with Aβ40, p<0.01for all the other data by Student's t test). A: Cortex. B: Hippocampus.

FIG. 21. Amyloid formation of hamster prion peptide PrP(108-144) in theabsence (panel A) or presence of V24P(10-40) (panel B). Solutions of 50μM PrP(108-144) with (panel B) or without (panel A) 50 μM V24P(10-40)were incubated in 20 mM NaOAc, pH 3.7/140 mM NaCl, at room temperaturefor different times. Samples were then removed and amyloid formation wasmeasured using the ThT binding assay.

FIG. 22. Effect of V24P(10-40)-PEI on Aβ peptide levels. APP/PS1 micewere treated from the age of 4 months to 8 months with PEI (control) orV24P(10-40)-PEI as described in the Examples. The levels of Aβ40 andAβ42 levels in the hippocampus and cortex were measured by ELISA. Thedata were presented as the mean ±standard deviation for 3 mice per group(*p<0.05; **p<0.01; ***p<0.001, Student's t test).

FIG. 23. V24P(10-40)-PEI decreases Aβ plaque accumulation in APP/PS1mice. Panel A: Representative microPET images of APP/PS1 mice takenafter treatment with either PEI (control) or V24P(10-40)-PEI for 8months. A microPET image of a wild type mouse (WT) is included forcomparison. Panel B: Quantitative analysis of [¹¹C]PiB uptake in thecortex, hippocampus, amydala, and olfactory bulb. The data are presentedas the mean ±standard deviation for 6 mice per group (*p<0.05; **p<0.01;***p<0.001, Student's t test).

DETAILED DESCRIPTION OF THE INVENTION

Neurodegenerative diseases are a group of neurological disorderscharacterized by a gradual loss of neurons in association with theformation of hallmark inclusion bodies (IBs), which results indysfunction of the nervous system and the eventual demise of patients.Takalo et al., Am J Neuro Degener Dis (2013) 2:1-14. Neurodegenerativediseases such as Huntington's disease (HD), Alzheimer's disease (AD),Parkinson's disease (PD), and others are considered as diseases mediatedby protein misfolding because of the formation of hallmark inclusionbodies (IBs). Yates, Nature Reviews (2012) 11: 352-353.

For instance, HD is an autosomal dominantly-inherited neurodegenerativedisease caused by an expansion of the CAG trinucleotide repeats in thegene Huntingtin (HTT), which encodes a mutant Htt (mHtt) proteincontaining a prolonged polyglutamine (polyQ) stretch. Zheng et al.,Progress in Mol Biol and Translational Sci (2012) 107:189-214. Althoughhaving a seemingly simple etiology, HD in fact, is very complex in itspathogenesis (Li et al., Mol Neurodengener (2006) 1:19) which involves alarge number of important biological processes and signaling pathwaysthat have gone awry. Munoz-Sanjuan et al., J Clin Invest (2011)121:476-83. Currently, no disease-modifying treatment for HD or otherneurodegenerative diseases is available. Thus, finding an effectivetherapeutic regimen is a focus of the neurodegenerative diseasecommunity. The aberrant pathways or processes may serve as valuabletherapeutic targets (Munoz-Sanjuan et al., J Clin Invest (2011)121:476-83); however, attempts to target these processes would produceundesired side effects as illustrated in the Semagacestat clinical trialfor AD. Doody et al., N Eng J Med (2013) 369:341-50.

Alzheimer's disease (AD), pathologically defined by the amyloid plaquesand neurofibrillary tangles (Nelson et al., J Neuropathol Exp Neurol(2012) 71:362-81), is the most common neurodegenerative disease thatcauses dementia across multiple cognitive domains, and its incidenceincreases exponentially among people >65 years of age. Reitz et al., NatRev Neurol (2011) 7:137-52. Despite the remarkable scientificadvancements and the huge amount of resources invested in drugdevelopment based on these discoveries, no effective disease-modifyingtherapy is currently available for AD. Castellani et al., BiochemPharmacol (2014) 88:671-6. Thus, it is one of the major unmet medicalneeds worldwide.

Although the etiology of AD remains unclear, alterations in multipleprocesses have been proposed as important causes and/or contributors,including the amyloid cascade hypothesis. Yamashima Prog Neurobiol(2013) 105:1-23; Swerdlow et al., Biochim Biophys Acta (2014)1842:1219-31; Erickson et al., J Cereb Blood Flow Metab (2013)33:1500-13; Clavaguera et al., Neuropharmacol (2014) 76 Pt A:9-15;Castello et al., Ageing Res Rev (2013) 12:282-8; Sutherland et al.,Redox Rep (2013) 18:134-41; Tiiman et al., Neurochem Int (2013)62:367-78; Puglielli Neurobiol Aging (2008) 29:795-811; Hardy, JAlzheimer's Dis (2006) 9:151-3; and Checler et al., J Neurochem (2012)120 Suppl 1:iii-iv. The amyloid cascade hypothesis proposes that“amyloid β-protein” (Aβ), a peptide of different lengths (39-43 aminoacids) with variations and modifications at both termini, plays acentral and initiative role in AD pathogenesis and/or progression. Hardyet al., Science (1992) 256:184-5; Hardy et al., Science (2002)297:353-6; Tanzi et al., Cell (2005) 120:545-55. Aβ is derived fromamyloid precursor protein (APP). During normal or non-amyloidogeniccatabolism, APP is cleaved by α- and γ-secretases, while, inamyloidogenic catabolism, it is cleaved by β- and γ-secretases. Thedifference in length of the Aβ peptide is partially due to the variancein the cutting sites of γ-secretase. Aβ peptides tend to self-aggregateinto amyloid fibrils and more cytotoxic oligomers. Walsh et al., JNeurochem (2007) 101:1172-84. Aβ40 and Aβ42 are two main species of Aβpeptides recovered from amyloid plaques with the latter being more proneto aggregate and cytotoxic.

Amyloid plaques belong to a large family of inclusion bodies (IBs),which are characteristics of a variety of neurodegenerative diseases,including Parkinson's disease (PD), Huntington's disease (HD), andamyotrophic lateral sclerosis (ALS). In spite of the differences in theconstituent proteins and complexity of the assembly mechanism, theco-existence of different neurodegenerative diseases and associated IBsis well-recognized in a substantial subset of patient cohorts.Keith-Rokosh Can J Neurol Sci (2008) 35:602-8; Tada et al., ActaNeuropathol (2012) 124:749-60; Schwab et al., J Neuropathol Exp Neurol(2008) 67:1159-65; Amador-Ortiz et al., Ann Neurol (2007) 61:435-45;Arai et al., Acta Neuropathol (2009) 117:125-36; Szpak et al., FoliaNeuropathol (2001) 39:63-71. In addition, abundant evidence demonstratesthat misfolded proteins from different diseases can cross-seed eachother to co-aggregate. Jucker et al., Ann Neurol (2011) 70:532-40; Ma etal., J Mol Biol (2012) 421:172-84; Guo et al., Cell (2013) 154:103-17;Waxman et al., J Neurosci (2011) 31:7601-18; Vitrenko et al., J BiolChem (2007) 282:1779-87; Wasmer et al., J Mol Biol (2010) 402:311-25;Yan et al., Am J Pathol (2007) 171:172-80. These findings suggest thatthe formation of IBs may be governed, at least in part, by a common setof thermodynamic principles (Jucker et al., Ann Neurol (2011)70:532-40), and interference with the association pathway toward themost thermodynamically stable oligomers or fibrils sheds light onamyloidosis therapy.

Conceptually, reducing the toxic species (the abnormal aggregates)formed by the misfolded protein or its derivative may be a practical andsafe therapeutic strategy. Mielcarek et al., PLoS Biol (2013)11:e1001717; Appl et al., Drug Discovery Today (2012) 17:1217-23. In HD,the prolonged polyQ confers an aberrant propensity for self-aggregationto form neurotoxic species to the mHtt protein. Oligomer or largefibrillary aggregates in nuclei and processes of neurons and glia arelinked with HD pathogenesis. Olshina et al., J Biol Chem (2010)285:21807-16; Ren et al., Nat Cell Biol (2009) 11:219-25; Hoffner etal., Prion (2007) 1:26-31; Marcellin et al., PLoS One (2012) 7:e44457;and Legleiter et al., J Biol Chem (2010) 285:14777-90. Indeed, thisapproach has been tested in several neurodegenerative disease modelsincluding HD (Kordasiewicz et al., Neuron (2012) 74:1031-44; Sontag etal., J Neurosci (2012) 32:11109-19) and AD (Aisen et al., CurrentAlzheimer Research (2007) 4:473-78; Frisardi et al., Current AlzheimerResearch (2010) 7:40-55) with encouraging results.

Since the formation of toxic misfolded proteins/derivatives is governedby a common set of thermophysical laws across different diseases, acommon strategy as described herein be used to develop therapeutic toolsby reducing the formation of the misfolded species in theseneurodegenerative diseases.

Accordingly, described herein is a rational design of therapeuticpeptides that are expected to reverse the pathogenic process of IBformation in a neurodegenerative disease as those described herein.Here, novel bipartite molecules containing at least one affinity moduleand at least one charged module were designed and their efficacy wasdemonstrated using cellular, APP/PS1 AD models, and R6/2 HD models. Thebipartite molecules would reduce toxic abnormal protein aggregatesacross various neurodegenerative diseases. The rational design is builton a principle of modular assembly of an affinity portion (e.g., apeptide affinity moiety) and at least one charged moiety, e.g.,positively charged or negatively charged. Without being bound by theory,the affinity moiety would facilitate binding of such therapeuticmolecules (bipartite molecules) to a protein component of an abnormalprotein aggregate or a monomer of the abnormal protein aggregate and thecharged portion(s) would prevent or reduce the formation of the abnormalprotein aggregate by, e.g., the repulsion force of charges (FIG. 1).

The approach described herein possesses several unique features andadvantages as compared with current therapeutic approaches forneurodegenerative diseases. Some examples are provided below. First, theaffinity moiety, which may be taken from the pathogenic peptide/proteinforming the abnormal aggregate not only significantly reduces laboriouswork finding and optimizing suitable peptide sequences, but alsoguarantees high affinity with IBs through its self-aggregating property.Second, the multiple charges in the charged moiety (e.g., a polyRfragment) render the bipartite molecules described herein (a) soluble inan aqueous environment, thereby simplifying the synthesis and deliveryprocesses and (b) cell-penetrable (Mitchell et al., J Pept Res (2000)56:318-25), making them suitable for inhibiting both extracellular andintracellular IB formation. Further, the charged moiety in the bipartitemolecules can prevent or reduce IB formation by charge repulsion afterbinding of the bipartite molecules to the pathogenic peptide/protein orprotein aggregate. Third, the combination of the charged moiety with theaffinity moiety provides great feasibility and flexibility in applyingthis design across different IB-containing diseases.

Accordingly, the present disclosure provides bipartite moleculescomprising an affinity moiety that binds an abnormal protein aggregateassociated with a disease or a component thereof (e.g., a proteinmonomer that forms the abnormal protein aggregate) and at least onecharged moiety, and uses of such bipartite molecules in preventing orreducing the formation of the abnormal protein aggregates and/or intreating diseases (e.g., neurodegenerative diseases) involving suchabnormal protein aggregates.

I. Bipartite Molecules

The bipartite molecule described herein comprise (i) a peptide affinitymoiety, which is capable of binding to an abnormal protein aggregate, ora component of that abnormal aggregate, and (ii) at least one chargedmoiety (e.g., positively charged or negatively charged), whichfacilitates the bipartite molecule to cross cell membranes and preventor decrease formation of the abnormal protein aggregation associatedwith a disease, such as a neurodegenerative disease as those describedherein.

(i) Peptide affinity moiety

The peptide affinity moiety of the bipartite molecule can be anypeptide-based (e.g., comprising a peptide) molecule capable of bindingto a targeted abnormal protein aggregates or a protein component thereof(e.g., a monomer capable of forming the abnormal protein aggregates or afragment thereof which is involved in the aggregate formation). Thepeptide affinity moiety may contain a peptide having up to 100 (e.g.,80, 60, 50, 40, 30, 20, or 10) amino acid residues, which can containeither naturally-occurring amino acids or modified ones such as D-aminoacids, or a combination thereof. In some examples, the peptide affinitymoiety may contain a peptide having 5-10, 5-20, 5-30, 10-15, 10-20,10-30, or 20-30 amino acid residues. The peptide affinity moiety may bemethylated or acetylated at the N-terminus, amidated at the C-terminus,or both to enhance stability.

Disease-associated abnormal protein aggregates and the correspondingprotein constituents are known in the art. Some examples are provided inTable 1 below:

TABLE 1 Exemplary Neurodegenerative Diseases and Abnormal ProteinAggregates Associated with such Diseases Exemplary Diseases involvingabnormal protein Aggregate Associated protein aggregates type componentsAlzheimer's disease [AD] amyloid amyloid precursor plaques, protein,amyloid β, presenilin neurofibrillary 1&2, tau, neurofilament tanglesprotein, alpha B-crystallin, transthyretin Huntington's disease [HD]inclusion uintingtin protein, bodies expanded polyglutamine tract inhuntingtin protein, alpha B- crystallin Parkinson's disease [PD] Lewyalpha-synuclein, bodies ubiquitin, neurofilament protein, alphaB-crystallin Dementia with Lewy body Lewy alpha-synuclein, [DLB] bodiesubiquitin Amyotrophic lateral sclerosis inclusion SOD-1, TAR DNA [ALS]bodies binding protein (TDP-43, or TARDBP), FUS, ubiquitin,neurofilament protein Frontotemporal lobar inclusion ubiquitin, TDP-43degeneration [FTLD]-TDP-43 bodies proteinopathy Frontotemporal lobarPick tau, fused in sarcoma degeneration [FTLD]-Tauopathy bodies (FUS),alpha B-crystallin Cortical basal degeneration Astroglial tau [CBD]inclusions Progressive supranuclear palsy neurofibrillary tau, Tau H1halotype [PSP] tangles, Lewy bodies Frontotemporal dementia and Lewymicrotubule-associated parkinsonism linked to chromosome 17 bodiesprotein tau [MAPT], tau [FTDP-17] Creutzfeldt-Jacob disease Aggregatesprion protein (PRNP), [CJD] and prion in the Scrapie form spongiform(PrP^(SC)) change

In some embodiments, the peptide affinity moiety may contain an aminoacid sequence derived either from a disease protein, which forms theabnormal protein aggregate or from the region of other proteins thatinteract with the disease protein or the abnormal protein aggregateformed thereby. In some embodiments, the peptide affinity moietycomprises a fragment of the disease protein (e.g., any of those listedin Table 1 above) that is involved in self-aggregation (formation of theabnormal protein aggregates).

In some examples, the peptide affinity moiety comprises a polyQfragment. Such a peptide affinity moiety can bind to and reduce/disruptformation of protein aggregates involving polyQ, for example, theaggregation of mHtt involved in HD. The polyQ moiety may comprise atleast 2, at least 5, at least 10, at least 15, at least 20 at least 25,at least 30, or at least 50 glutamine (Q) amino acid residues (e.g.,5-10, 5-15, 5-20, 5-30, or 10-20 Q residues). In some embodiments thepeptide affinity moiety is 5Q, 10Q, 15Q, or 20Q.

The protein affinity moiety may contain a fragment derived from awild-type disease protein (as those listed in Tables 1, 3, and 4 anddescribed herein) or from a mutant form or modified form of the diseaseprotein, particularly those involved in disease pathogenesis. Themodified form of the protein may include post translationalmodifications such as phosphorylation. For example, neurofibrillarytangles (NFTs) may be formed by hyperphosphorylation of themicrotubule-associated protein Tau. Accordingly, the peptide affinitymoiety may contain the hyperphosphorylated form, or a portion of thehyperphosphorylated form of Tau. Hyperphosphorylation may be achieved byemploying the use of various known kinases or by phosphomimetic aminoacid substitutions that mimic a phosphorylated protein. For example,aspartic acid (D) is chemically similar to phospho-serine. Therefore,when aspartic acid replaces a serine, it is a phosphomimetic ofphospho-serine.

In some embodiments, the peptide affinity moiety may contain a fragmentderived from amyloid β-protein, SOD1, Tau, TDP-43, α-synuclein,ubiquitin, neurofilament protein, alpha B crystalline, PrP^(SC), ortransthyretin. Such a fragment may involve in formation of proteinaggregate containing the disease protein. For example, fragment Aβ25-35from amyloid β-protein, which may contain an N-methylated Gly33, caninteract with amyloid plaques. Hughes et al., J Biol Chem (2000)275:25109-115. In another example, an Aβ40 mutant peptide, V24P, withthe V24 replaced by D-form proline (^(D)P), remains as random coils inbuffer and forms amorphous aggregates at a high peptide concentration.Chang et al., J Mol Biol (2009) 385:1257-65. Mixing V24P with Aβ40 at a1:1 molar ratio inhibits amyloid formation and the cytotoxicity of Aβ40.Iwata et al., Pharmacol Therapeut (2005) 108:129-48.

In some examples, the peptide affinity moiety may comprise the aminoacid sequence GSNKGAIIGLM (SEQ ID NO: 1), which is derived from amyloidβ-protein. Alternatively, the affinity moiety may comprise the aminoacid sequence YEVHHQKLVFFAED^(D)PGSNKGAIIGLMVGGVV (SEQ ID NO: 2), inwhich ^(D)P refers to the D-form of proline. In another example, theaffinity moiety may comprise RPRTRLHTHRNR (SEQ ID NO: 8), which may bean Aβ42-binding 12-mer D-form peptide as described in Wiesehan et al.(ChemBioChem (2003) 4:748-53), the relevant teachings therein areincorporated by reference herein. Other exemplary amyloid β-proteinfragments for use as the peptide affinity moiety in making the bipartitemolecules described herein are provided in Table 3 below.

Any of the peptide affinity moieties as described herein can be preparedby, e.g., chemical synthesis or recombinant technology.

(ii) Charged Moiety

The charged moiety of the bipartite molecule may prevent or reduce theformation of abnormal protein aggregates by misfolded disease proteinsor may lead to dissociation of existing abnormal protein aggregates bythe force of charge repulsion. See, e.g., FIG. 1. Further, the chargedmoiety may facilitate the bipartite molecule containing such to crosscell membranes and/or blood brain barrier (BBB).

In some embodiments, the charged moiety may contain a peptide comprisingup to 100 (e.g., 80, 60, 50, 40, 30, 20, or 10) amino acid residues,which may contain at least 2, at least 4, at least 5, at least 8, atleast 10, at least 15, at least 20, at least 25, or at least 30 chargedamino acids. In some examples, the charged moiety may be a peptidehaving all charged amino acid residues. For example, the peptide maycontain at least one (e.g., at least 2, 4, 5 or 10) negatively chargedamino acid, e.g., aspartic acid (e.g., a polyD fragment) or glutamicacid (e.g., a polyE fragment), or a combination of D and E amino acids.Alternatively, the peptide may contain at least one (e.g., at least 2,4, 5, or 10) positively charged amino acid, e.g., arginine, histidine,lysine, or a combination thereof. In some examples, the charged moietycontains a stretch of arginine (e.g., a polyR fragment) or lysineresidues (e.g., a polyK fragment). As used herein, a polyR, polyK,polyD, or polyE fragment refers to a stretch of R, K, D, or E residues.Such a fragment may contain up to 30 (e.g., 25, 20, 15, 10 or 5) R, K,D, or E residues. In some examples, the charged moiety may be a peptidecontaining a combination of different negatively charges amino acids ora combination of different positively charged amino acids.

In addition to preventing the misfolded target protein from formingabnormal protein aggregates or to dissociate it from existingaggregates, a charged moiety described herein, for example, a polyRfragment, can facilitate a bipartite molecule comprising such topenetrate through cell membranes, the blood brain barrier (BBB), orboth. In some embodiments, the charged moiety has the amino acidsequence RRRRRRRR (SEQ ID NO: 9).

In some embodiments, the charged moiety can be a cell-penetratingpeptide (CPP) such as TAT, referring to a peptide comprising the aminoacid sequence of GRKKRRQRRRPQ (SEQ ID NO: 10), which is derived from thetransactivator of transcription (TAT) of human immunodeficiency virus.Cell-penetrating peptides (CPPs) have been used to overcome thelipophilic barrier of the cellular membranes and deliver both largemolecules and even small particles (e.g., proteins, DNA, antibodies,contrast (imaging) agents, toxins, and nanoparticular drug carriersincluding liposomes) inside the cell for their biological actions. Apeptide-based charged moiety may be prepared by chemical synthesis orrecombinant technology.

In other embodiments, the charged moiety may be a non-peptide polymer,such as a polyethylenimine (PEI) molecule. Methods for synthesizingPEI-conjugated peptides are well known in the art. For example, PEIconjugated peptides may be synthesized by the batchfluorenylmethoxycarbonyl (fmoc)-polyamide method.

(iii) Configurations of the Affinity and Charged Moieties in theBipartite Molecules

In any of the bipartite molecules as described herein, the peptideaffinity moiety and the charged moiety or moieties are linked (e.g.,covalently) and may be configured in a suitable manner. In someembodiments, the bipartite molecule contains a peptide affinity moietyas described herein and a single charged moiety. The charged moiety canbe attached to either the N-terminus or the C-terminus of the peptideaffinity moiety, either directly or via a suitable linker. If thecharged moiety is also peptide based, the peptide affinity moiety andthe charged peptide may be linked via a peptide bond. If the chargedmoiety is a non-peptide polymer, such as PEI, the non-peptide polymermay be linked to either the N-terminus or the C-terminus of the peptideaffinity moiety via a suitable bond (e.g., a suitable covalent bond).

In some embodiments, the bipartite molecule may contain two chargedmoieties, which can be either identical or different. In other examples,the two charged moieties are both polyR fragments. For example, apoly-arginine (polyR) moiety having eight arginine residues is linked tothe amino-terminus of the peptide affinity moiety and anotherpoly-arginine (polyR) moiety having eight arginine residues can belinked to the carboxy-terminus of the peptide affinity moiety.

In some embodiments, the two charged moieties can be different. Forexample, one of the charged moieties can be a peptide-based moiety suchas a polyR fragment and the other charged moieties can be a non-peptidepolymer such as PEI. The polyR fragment and PEI may be linked to theN-terminus and C-terminus of the peptide affinity moiety, respectively,or vice versa. In some embodiments the two charged moieties may haveopposing charges. For example, a positively charged poly-arginine(polyR) moiety may be linked to the amino-terminus and a negativelycharged poly-aspartate (polyD) moiety may be linked to thecarboxy-terminus of the peptide affinity moiety. In some examples, thecharged moieties may both carry a positive charge or both carry anegative charge. For example, a positively charged poly-arginine (polyR)moiety may be linked to the amino-terminus and a positively chargedpoly-lysine (polyK) moiety may be linked to the carboxy-terminus of thepeptide affinity moiety. In some embodiments the bipartite molecule isRRRRRRRRGSNKGAIIGLM-PEI (SEQ ID NO: 4). It should be appreciated thatthe charged moiety, or moieties, of the bipartite molecule, as describedherein, may be configured in any number of ways with respect to thepeptide affinity moiety.

In some examples, the peptide moiety or moieties may be modified at theC-terminus (e.g., acetylation), the N-terminus (e.g., methylation), orboth for at least enhancing stability of the bipartite moleculecontaining such.

II. Pharmaceutical Compositions

The bipartite molecules described herein can be mixed with apharmaceutically acceptable carrier (excipient) to form a pharmaceuticalcomposition for use in treating a target disease. “Acceptable” meansthat the carrier must be compatible with the active ingredient of thecomposition (and preferably, capable of stabilizing the activeingredient) and not deleterious to the subject to be treated.Pharmaceutically acceptable excipients (carriers) including buffers,which are well known in the art. See, e.g., Remington: The Science andPractice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins,Ed. K. E. Hoover.

The pharmaceutical compositions to be used in the present methods cancomprise pharmaceutically acceptable carriers, excipients, orstabilizers in the form of lyophilized formulations or aqueoussolutions. (Remington: The Science and Practice of Pharmacy 20th Ed.(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations used, and may comprise buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In some examples, the pharmaceutical composition described hereincomprises liposomes containing the bipartite molecules (or the encodingnucleic acids) which can be prepared by methods known in the art, suchas described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688(1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); andU.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Particularlyuseful liposomes can be generated by the reverse phase evaporationmethod with a lipid composition comprising phosphatidylcholine,cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter.

The bipartite molecules may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are known in theart, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the bipartite molecule, which matricesare in the form of shaped articles, e.g., films, or microcapsules.Examples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(v nylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeutic bipartitemolecule compositions may be placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosageforms such as tablets, pills, capsules, powders, granules, solutions orsuspensions, or suppositories, for oral, parenteral or rectaladministration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient can be mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 .im, particularly 0.1 and 0.5 .im, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing bipartitemolecules with Intralipid™ or the components thereof (soybean oil, eggphospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas set out above. In some embodiments, the compositions are administeredby the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

III. Methods of Treatment

Any of the bipartite molecules are useful in preventing/reducing theformation of protein aggregates associated with a disease such as aneurodegenerative disease as described herein, or disrupting such aprotein aggregate. The bipartite molecule can also be used for treatinga disease or disorder, particularly a neurodegenerative disease ordisorder, characterized by abnormal protein aggregates.

To practice the method disclosed herein, an effective amount of thepharmaceutical composition described herein can be administered to asubject (e.g., a human) in need of the treatment via a suitable route,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, inhalation or topical routes. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution. Alternatively, the bipartite molecules as describedherein can be aerosolized using a fluorocarbon formulation and a metereddose inhaler, or inhaled as a lyophilized and milled powder. In oneexample, the bipartite molecule is administered via an intranasal route,e.g., by nasal drops.

The subject to be treated by the methods described herein can be amammal, more preferably a human. Mammals include, but are not limitedto, farm animals, sport animals, pets, primates, horses, dogs, cats,mice and rats. A human subject who needs the treatment may be a humanpatient having, at risk for, or suspected of having a targetdisease/disorder, such as Alzheimer's disease (AD), Huntington's disease(HD), Parkinson's disease (PD), dementia with Lewy bodies (DLB),amyotrophic lateral sclerosis (ALS), frontotemporal lobardegeneration-TDP-43 proteinopathy, frontotemporal lobardegeneration-tauopathy (Pick's disease, cortical basal degeneration,progressive supranuclear palsy, FTDP-17), Creutzfeldt-Jacob disease(CJD), or another disease. A subject having a target disease or disordercan be identified by routine medical examination, e.g., laboratorytests, organ functional tests, CT scans, or ultrasounds. A subjectsuspected of having any of such target disease/disorder might show oneor more symptoms of the disease/disorder. A subject at risk for thedisease/disorder can be a subject having one or more of the risk factorsfor that disease/disorder.

As used herein, “an effective amount” refers to the amount of eachactive agent required to confer therapeutic effect on the subject,either alone or in combination with one or more other active agents. Insome embodiments, the therapeutic effect is reduced abnormal proteinaggregates, or the prevention of abnormal protein aggregate formation.Determination of whether an amount of the bipartite molecule achievedthe therapeutic effect would be evident to one of skill in the art.Effective amounts vary, as recognized by those skilled in the art,depending on the particular condition being treated, the severity of thecondition, the individual patient parameters including age, physicalcondition, size, gender and weight, the duration of the treatment, thenature of concurrent therapy (if any), the specific route ofadministration and like factors within the knowledge and expertise ofthe health practitioner. These factors are well known to those ofordinary skill in the art and can be addressed with no more than routineexperimentation. It is generally preferred that a maximum dose of theindividual components or combinations thereof be used, that is, thehighest safe dose according to sound medical judgment.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. Frequency ofadministration may be determined and adjusted over the course oftherapy, and is generally, but not necessarily, based on treatmentand/or suppression and/or amelioration and/or delay of a targetdisease/disorder. Alternatively, sustained continuous releaseformulations of a bipartite molecule may be appropriate. Variousformulations and devices for achieving sustained release are known inthe art.

In one example, dosages for a bipartite molecule as described herein maybe determined empirically in individuals who have been given one or moreadministration(s) of the bipartite molecule. Individuals are givenincremental dosages of the antagonist. To assess efficacy of theantagonist, an indicator of the disease/disorder can be followed.

Generally, for administration of any of the bipartite moleculesdescribed herein, an initial candidate dosage can be about 2 mg/kg. Forthe purpose of the present disclosure, a typical daily dosage mightrange from about any of 0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to3 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on the factorsmentioned above. For repeated administrations over several days orlonger, depending on the condition, the treatment is sustained until adesired suppression of symptoms occurs or until sufficient therapeuticlevels are achieved to alleviate a target disease or disorder, or asymptom thereof. An exemplary dosing regimen comprises administering aninitial dose of about 2 mg/kg, followed by a weekly maintenance dose ofabout 1 mg/kg of the bipartite molecule, or followed by a maintenancedose of about 1 mg/kg every other week. However, other dosage regimensmay be useful, depending on the pattern of pharmacokinetic decay thatthe practitioner wishes to achieve. For example, dosing from one-fourtimes a week is contemplated. In some embodiments, dosing ranging fromabout 3 μg/mg to about 2 mg/kg (such as about 3 μg/mg, about 10 μg/mg,about 30 μg/mg, about 100 μg/mg, about 300 μg/mg, about 1 mg/kg, andabout 2 mg/kg) may be used. In some embodiments, dosing frequency isonce every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks;or once every month, every 2 months, or every 3 months, or longer. Theprogress of this therapy is easily monitored by conventional techniquesand assays. The dosing regimen (including the bipartite molecule used)can vary over time.

In some embodiments, for an adult patient of normal weight, dosesranging from about 0.3 to 5.00 mg/kg may be administered. The particulardosage regimen, i.e., dose, timing and repetition, will depend on theparticular individual and that individual's medical history, as well asthe properties of the individual agents (such as the half-life of theagent, and other considerations well known in the art).

For the purpose of the present disclosure, the appropriate dosage of abipartite molecule as described herein will depend on the specificbipartite molecule, bipartite molecules, the type and severity of thedisease/disorder, whether the bipartite molecule is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antagonist, and the discretion ofthe attending physician. A clinician may administer a bipartitemolecule, until a dosage is reached that achieves the desired result. Insome embodiments, the desired result is a decrease in abnormal proteinaggregates. Methods of determining whether a dosage resulted in thedesired result would be evident to one of skill in the art.

Administration of one or more bipartite molecules can be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of an bipartite molecule may be essentially continuousover a preselected period of time or may be in a series of spaced dose,e.g., either before, during, or after developing a target disease ordisorder. In some examples, the amount of the bipartite molecule iseffective in reducing the formation of an abnormal protein aggregate byat least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or above.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has a target disease or disorder, a symptom of thedisease/disorder, or a predisposition toward the disease/disorder, withthe purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect the disorder, the symptom of the disease,or the predisposition toward the disease or disorder.

Alleviating a target disease/disorder includes delaying the developmentor progression of the disease, or reducing disease severity. Alleviatingthe disease does not necessarily require curative results. As usedtherein, “delaying” the development of a target disease or disordermeans to defer, hinder, slow, retard, stabilize, and/or postponeprogression of the disease. This delay can be of varying lengths oftime, depending on the history of the disease and/or individuals beingtreated. A method that “delays” or alleviates the development of adisease, or delays the onset of the disease, is a method that reducesprobability of developing one or more symptoms of the disease in a giventime frame and/or reduces extent of the symptoms in a given time frame,when compared to not using the method. Such comparisons are typicallybased on clinical studies, using a number of subjects sufficient to givea statistically significant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of a target disease or disorder includes initialonset and/or recurrence.

In some embodiments, the bipartite molecules described herein areadministered to a subject in need of the treatment at an amountsufficient to reduce abnormal protein aggregates by at least 5% (e.g.,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. Inother embodiments, the bipartite molecules are administered in an amounteffective in reducing the activity level of a target antigens by atleast 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater).

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical composition tothe subject, depending upon the type of disease to be treated or thesite of the disease. This composition can also be administered via otherconventional routes, e.g., administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection or infusion techniques. Inaddition, it can be administered to the subject via injectable depotroutes of administration such as using 1-, 3-, or 6-month depotinjectable or biodegradable materials and methods. In some examples, thepharmaceutical compositions is administered intraocularlly orintravitreally.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethyformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water soluble bipartite molecules can beadministered by the drip method, whereby a pharmaceutical formulationcontaining the bipartite molecule and a physiologically acceptableexcipients is infused. Physiologically acceptable excipients mayinclude, for example, 5% dextrose, 0.9% saline, Ringer's solution orother suitable excipients. Intramuscular preparations, e.g., a sterileformulation of a suitable soluble salt form of the bipartite molecule,can be dissolved and administered in a pharmaceutical excipient such asWater-for-Injection, 0.9% saline, or 5% glucose solution.

In one embodiment, a bipartite molecule is administered viasite-specific or targeted local delivery techniques. Examples ofsite-specific or targeted local delivery techniques include variousimplantable depot sources of the bipartite molecule or local deliverycatheters, such as infusion catheters, an indwelling catheter, or aneedle catheter, synthetic grafts, adventitial wraps, shunts and stentsor other implantable devices, site specific carriers, direct injection,or direct application. See, e.g., PCT Publication No. WO 00/53211 andU.S. Pat. No. 5,981,568.

Targeted delivery of therapeutic compositions containing an antisensepolynucleotide, expression vector, or subgenomic polynucleotides canalso be used. Receptor-mediated DNA delivery techniques are describedin, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiouet al., Gene Therapeutics: Methods And Applications Of Direct GeneTransfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988)263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc.Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991)266:338.

Therapeutic compositions containing a polynucleotide (e.g., thoseencoding the bipartite molecules described herein) are administered in arange of about 100 ng to about 200 mg of DNA for local administration ina gene therapy protocol. In some embodiments, concentration ranges ofabout 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg toabout 500 _(l)ug, and about 20 μg to about 100 μg of DNA or more canalso be used during a gene therapy protocol.

In some examples, patients with or at risk for Huntington's disease maybe treated with a bipartite molecule having an affinity moiety thatcomprises polyQ attached to at least one charged moiety. In someembodiments the bipartite molecule is RRRRRRRRWDQQQQQQQQQQ (SEQ ID NO:6), or RRRRRRRRWDQQQQQQQQQQQQQQQ (SEQ ID NO: 7). Alternatively, apatient with or at risk for Alzheimer's disease may be treated with abipartite molecule having an affinity moiety that comprises a portion ofthe amyloid (3-protein attached to at least one charged moiety. In someembodiments the bipartite molecule is RRRRRRRRGSNKGAIIGLM (SEQ ID NO:3), RRRRRRRRGSNKGAIIGLM-PEI (SEQ ID NO: 4), orYEVHHQKLVFFAED^(D)PGSNKGAIIGLMVGGVV-PEI (SEQ ID NO: 5).

The particular dosage regimen, i.e., dose, timing and repetition, usedin the method described herein will depend on the particular subject andthat subject's medical history.

In some embodiments, more than one bipartite molecule, or a combinationof a bipartite molecule and another suitable therapeutic agent, may beadministered to a subject in need of the treatment. The bipartitemolecule can also be used in conjunction with other agents that serve toenhance and/or complement the effectiveness of the agents.

Treatment efficacy for a target disease/disorder can be assessed by,e.g., a method described in the Examples below.

IV. Kits for Use in Alleviating Neurodegenerative Diseases AssociatedAbnormal Protein Aggregates

The present disclosure also provides kits for use in alleviatingdiseases/disorders associated abnormal protein aggregates, such asAlzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease(PD), dementia with Lewy bodies (DLB), amyotrophic lateral sclerosis(ALS), frontotemporal lobar degeneration-TDP-43 proteinopathy,frontotemporal lobar degeneration-tauopathy (Pick's disease, corticalbasal degeneration, progressive supranuclear palsy, FTDP-17), orCreutzfeldt-Jacob disease (CJD). Such kits can include one or morecontainers comprising a bipartite molecule, e.g., any of those describedherein.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise a description of administration of thebipartite molecule to treat, delay the onset, or alleviate a targetdisease as those described herein. The kit may further comprise adescription of selecting an individual suitable for treatment based onidentifying whether that individual has the target disease. In stillother embodiments, the instructions comprise a description ofadministering a bipartite molecule to an individual at risk of thetarget disease.

The instructions relating to the use of a bipartite molecule generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating, delaying the onset and/or alleviating a disease or disorderassociated with the presence of abnormal protein aggregates, such asthose described herein. Instructions may be provided for practicing anyof the methods described herein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a bipartite molecule as those described herein.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the invention provides articles of manufacture comprisingcontents of the kits described above.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. The examples describedin this application are offered to illustrate the molecules,pharmaceutical compositions, and methods provided herein and are not tobe construed in any way as limiting their scope.

Example 1 Exemplary Bipartite Therapeutic Peptides and Uses thereof inTreating Huntington's Disease

Huntington's disease (HD) is caused by an expansion of the CAGtrinucleotide repeats in the Huntingtin (HTT) gene. The expanded CAGrepeats encode an elongated polyglutamine stretch (polyQ) within themutant Htt (mHtt) protein, which leads to the misfolding and aggregationof mHtt. Provided herein is a series of therapeutic peptides , forexample, 8R5Q, 8R10Q, 8R15Q and 8R20Q, which contains polyarginines(e.g., 8R) and a short stretch of polyQ (e.g., 5Q, 10Q, 15Q, or 20Q).The polyQ sequence was expected to confer a specific affinity for thetherapeutic peptides to bind to mHtt and the polyarginine fragmentpossesses the capability to penetrate neurons and prevent mHtt/peptideself-aggregation charge repulsion. This was demonstrated by theobservation that in Neuro2a cells (mouse neuroblastoma), 8R10Qco-localized with 109QmHtt aggregates. Both 8R10Q and 8R15Qsignificantly decreased the size of 109QmHtt aggregates. In addition,8R10Q reduced the level of 109QmHtt, which was blocked by the proteasomeinhibitor MG132, indicating that the peptide enhanced cellular abilityto degrade 109QmHtt. Functionally, 8R10Q attenuated the 109QmHtt-inducedtoxicity in the growth of undifferentiated Neuro2a cells by a MTT assayand the retinoic acid-mediated neurite outgrowth of Neuro2a cells. Invivo, 8R10Q significantly delayed the motor and memory deterioration ofR6/2 transgenic mice, a HD mouse model.

Histopathologically, 8R10Q also reduced the aggregation of mHtt andneuronal loss, and ameliorated the activation of astrocytes andmicroglia. Intriguingly, 8R10Q decreased the diabetic phenotype,inflammatory index, and abnormal liver function of R6/2 mice.Altogether, the therapeutic peptides designed by this modular principleworked against HD, which may also shed light on the development of atherapeutic strategy against other neurodegenerative diseases.

Design and Synthesis of the Bipartite Peptides

The rational design of therapeutic peptides to reduce the toxicmisfolded proteins/derivatives across different neurodegenerativediseases based on the principle of modular assembly was attempted. Theconjectured therapeutic peptides would assume a bipartite or tripartitestructure composed of a full or partial sequence from theself-aggregating region of the misfolded protein (e.g., mHtt or amyloidβ peptide, etc.) flanked either upstream or downstream (bipartite) orboth (tripartite) by a stretch of charged amino acids. The rationalebehind this design was that the self-aggregating sequence would providea specific affinity between the peptide and the misfoldedprotein/derivative through its self-aggregating property. The chargedamino acid chosen in the study may be arginine. Polyarginine stretchenabled the therapeutic peptides to penetrate through cell membrane intocytoplasmic and nuclear compartments (Mitchell, J Pept Res (2000)56:318-25). It was also expected to prevent therapeutic peptides fromself-aggregation. Moreover, polyarginine can prevent the mHtt/peptidehybrids from self-aggregation by charge repulsion force (FIG. 1).

In this study, the rational design was tested for HD. Several bipartitetherapeutic peptides containing 8 consecutive arginines (8R) attached toa stretch of consecutive glutamines (polyQ) were synthesized and theirbiological activity of decreasing the aggregation propensity of the mHttprotein was investigated. The length of the polyQ of these bipartitepeptides was 5 (8R5Q), 10 (8R10Q), 15 (8R15Q) or 20 (8R20Q) (Table 2).Peptides 8R5Q and 8R10Q were highly soluble in water or culture medium(Table 2), and remained unchanged in their retention time and amount forat least 28 days (FIG. 2, panels A and B), indicating these two peptideswere quite stable. In contrast, 8R15Q was partially soluble, but 8R20Qwas insoluble, and both underwent a time-dependent decrease in theretention time and amount (FIG. 2, panels A and B) during HPLC analysis,indicating their high stability. Thus, 8R10Q was selected as the leadpeptide for subsequent studies given its superior biochemicalproperties.

TABLE 2 The sequence and biochemical properties of therapeutic peptides.MALDI-TOF Solubility Peptide Sequence Formula Calculated Found(DMEM/10%FBS) 8R NH₂-(R)₈-W-D-CONH₂ C₆₃H₁₁₃N₃₅O₁₃ 1567.8 1568.0 Soluble8R5Q NH₂-(R)₈-W-D-(Q)₅-CONH₂ C₈₈H₁₅₃N₄₅O₂₃ 2208.8 2208.5 Soluble 8R10QNH₂-(R)₈-W-D-(Q)₁₀-CONH₂ C₁₁₃H₁₉₃N₅₅O₃₃ 2849.2 2849.2 Soluble 8R15QNH₂-(R)₈-W-D-(Q)₁₅-CONH₂ C₁₃₈H₂₃₃N₆₅O₄₃ 3489.8 3489.8 aggregated 8R20QNH₂-(R)₈-W-D-(Q)₂₀-CONH₂ C₁₆₃H₂₇₃N₇₅O₅₃ 4130.5 4130.5 aggregated s8R10QNH₂- C₁₁₃H₁₉₃N₅₅O₃₃ 2849.2 2849.5 Soluble RQQRRQQDQRQWQRQRQQRR- CONH₂TAMRA- TAMRA-NH-(R)₈-W-D-CONH₂ C₈₈H₁₃₈N₃₇O₁₇ 1982.3 1981.0 Soluble 8RWDTAMRA- TAMRA-NH-(R)₈-W-D- C₁₃₈H₂₁₈N₅₇O₃₇ 3264.7 3264.6 Soluble 8R10Q(Q)₁₀-CONH₂ TAMRA- TAMRA-NH- C₁₃₈H₂₁₈N₅₇O₃₇ 3264.7 3264.6 Soluble s8R10QRQQRRQQDQRQWQRQRQQRR- CONH₂s8R10Q: scrambled 8R10Q

The sequence in Table 2, from top to bottom, correspond to SEQ ID NOs:11-19.

Decreasing mHtt Aggregation by PolyR-PolyQ Peptide

The ability of 8R10Q to bind to the 109QmHtt was examined by determiningits co-localization with the 109QmHtt aggregates. The 8R10Q peptide waslabeled with carboxytetramethylrhodamine (TAMRA) dye (FIG. 3, panel A),and added into Neuro2a cells expressing 25QHtt or 109QmHtt. The TAMRAdye alone failed to associate with the 109QmHtt aggregates, and thelabeled 8R10Q also failed to conform to the pattern of 25QHtt. Incontrast, the TAMRA-labeled 8R10Q peptide was co-localized with theaggregates (FIG. 3, panel B). These results clearly validated theability of 8R10Q to interact with the mHtt, as expected.

To probe into the details of the effect of peptide on the mHttaggregates, the parameters of the mHtt aggregates were visualized andmeasured using the Total Internal Reflection Fluorescence (TIRF)microscopy. As shown in FIG. 3, panel C, large solid 109QmHtt aggregateswere readily observed in the Neuro2a cells treated with water, 8R, or ascrambled peptide (s8R10Q); however, mainly small punctate aggregateswere seen in cells treated with 8R10Q, which remained so throughout thetested time points. Quantitative data showed that the average size ofthe aggregates from water, 8R or scrambled peptide-treated cells were3.6 μm², 3.3 μm², 1.7 μm², respectively; while those from the 8R10Q- or8R15Q-treated cells were dramatically reduced to 0.3 μm² and 0.2 μm²,respectively (FIG. 3, panel D). Notably, a subset of the aggregates inthe former control groups were larger than 10 μm², which were neveridentified in the latter. The number of aggregates in 20 cells was alsocalculated. 8R10Q and 8R15Q increased the number of the aggregates by4-5 fold (FIG. 3, panel E). These results suggested that 8R10Q or 8R15Qprevented the small punctate structures from forming large conglomeratesof 109QmHtt.

The small aggregates were tracked for 24 hours using live cell imaging.As expected, multiple small punctuates within one cell fused with eachother with time, and eventually formed one dominant or several largeaggregates. 8R10Q or 8R15Q treatment rendered these punctuates unchangedthroughout the observation time period.

Whether the peptides could modulate the aggregation propensity of mHttwas next investigated. As shown in FIG. 4, panels A and C, 20 μM ofpeptide 8R10Q or 8R15Q, added at 8 hours (T1) or 8 plus 24 hours (T1+T2)after transfection significantly reduced the aggregated 109QmHtt ascompared with water or the 8R peptide control by filter trap assay.However, when the peptide was added at 24 hours alone, no obviouseffects were observed. These results showed that 8R10Q and 8R15Qpeptides effectively prevented the mHtt from aggregation.

For further characterization, Neuro2a lysates were separated into theRIPA-soluble (sol) and insoluble (ins) fractions (FIG. 4, panel B).8R10Q peptide decreased the level of mHtt in both the soluble andinsoluble fractions as compared with water, 8R, or the scrambled(s8R10Q) control (FIG. 4, panel D). Since the ubiquitin proteasomalsystem (UPS) had been previously shown to be important for mHttdegradation (Martin-Aparicio, J Neurosci (2001) 21:8772-81; Jana, HumMol Genet (2001) 10:1049-59), MG132, an inhibitor of the UPS, was usedto block the peptide-induced decrease in the mHtt. MG132 reversed thelevel of the 109QmHtt in the peptide group (FIG. 4, panel E). Aquantitative assay showed that MG132 treatment increased the level ofmHtt across all groups, indicating a basal turnover of 109QmHtt by UPS.However, the ratio of the MG132:DMS0 109QmHtt was much higher for the8R10Q treated samples (FIG. 4, panel F). These results indicated thatthe peptide reduced the mHtt through enhancement of its degradation byUPS.

Protection of Cells Against the Neurotoxicity of 109QmHtt and H₂O₂ byBipartite Peptides

It has long been reported that HD patients have higher levels ofoxidative stress linked with mitochondrial dysfunction induced by mHtt.Browne, Brain Pathol (1999) 9:147-63; and Tasset, Revista de Neurologia(2009) 49:424-9. mHtt and oxidative stress may form a self-reinforcingvicious cycle in HD. In the present study, approximately 83% of Neuro2acells expressing 25QHtt treated with 50 1μM H₂O₂ with or withoutscrambled or bipartite therapeutic peptides survived (FIG. 5, panel A).On the other hand, the survival rate of Neuro2a cells expressing109QmHtt decreased to ˜72%, indicating that the 109QmHtt rendered cellsmore vulnerable to the oxidative stress of H₂O₂. 8R10Q or 8R15Qtreatment increased the survival rate to levels above 80% (FIG. 5, panelA).

The expression of 109QmHtt reduced the growth of Neuro2a cells, and sodid the lower levels of H₂O₂ (12.5 μM) (FIG. 5, panel B). Peptide 8R10Qameliorated the reduction in growth induced not only by the toxicity of109QmHtt, but also by H₂O₂, as compared with 8R or the scrambledcontrol.

Retinoic acid (RA) induced the differentiation of Neuro2a cells withneurite outgrowth. 109QmHtt decreased the number of cells bearingRA-induced neurites relative to 25QHtt (FIG. 5, panels C and D). 8R10Qtreatment significantly reversed the defect in neurite outgrowth causedby 109QmHtt as compared with water or the scrambled control. The 8Rpeptide also rescued the cells from this toxicity to some extent, butthe results were not statistically significant. These results clearlyshowed that the bipartite peptides could protect Neuro2a cells againstthe toxicity caused either by 109QmHtt and/or by H₂O₂.

Therapeutic Effect of 8R10Q on R6/2 Transgenic Mice

To test the therapeutic effect of the peptide in vivo, the 8R10Q peptidewas delivered 6 days/week into the wild type (WT) or R6/2 transgenicmice through intranasal aspiration or continuously with the Alzetosmotic minipumps into the neostriatum. The body weights of the WT orR6/2 mice treated with 8R10Q through either route remained comparable tothose treated with PBS. As shown in FIG. 6, panel A, the motordeterioration assessed by the rotarod of R6/2 mice became significantfrom 11 weeks of age, but the 8R10Q treatment effectively delayed thephenotype until 13 weeks. In fact, the 8R10Q-treated R6/2 mice performedeven better than the 11 week-old PBS-treated mice on average. The 8R10Qpeptide delivered intracerebrally exhibited a very similar therapeuticeffect to that given intranasally. These results showed thatadministration route does not affect this effect. In addition,intranasal 8R10Q also corrected the memory deficit of 13 week-old R6/2mice as shown in the T maze test (FIG. 6, panel B). The R6/2 mice werepreviously reported to have diabetes. Bjorkqvist et al., Hum Mol Genet(2005) 14:565-74; and Hunt et al., Experimental Brain Res (2005)166:220-9. The intranasal administration of 8R10Q peptide delayed therise and decreased the level of blood sugar (FIG. 6, panel C).Similarly, 8R10Q given intracerebrally also significantly corrected thediabetes phenotype. Lastly, all R6/2 mice treated with PBS died beforeor at 16 weeks of age; however, 8R10Q given intranasally preventedpremature death by 41% at 16 weeks of age (FIG. 6, panel D). To sum up,the 8R10Q peptide effectively delayed the onset of disease andameliorated the pathological phenotypes.

Decrease of Neuropathology by 8R10Q Peptide

R6/2 transgenic mice exhibited a decrease in cortical thickness due tothe loss of cortical neurons. Interestingly, 8R10Q peptide preventedcortical thinning (FIG. 7, panels A and B), suggesting that this peptidecould rescue neurons from death. Indeed, the neuronal count revealedthat the loss of neurons in both the cortex (FIG. 7, panels C and D) andstriatum (FIG. 7, panels E and F) of the R6/2 mice was significantlyprevented by the intranasal 8R10Q treatment.

To correlate the beneficial effect with the aggregates of mHtt,immunohistochemistry was performed to calculate the areas occupied bythe aggregates per unit area (150 μm²). The method was selected becausethe frequent clustering of the aggregates into conglomerates rendered anaccurate count of the aggregate number very difficult. As shown in FIG.8, panels A and B, the 8R10Q peptide decreased the areas of aggregatesby ˜60% in both the cortex and striatum as compared with the PBScontrol. In addition, the intensity which represented the amount of mHttin the aggregates was also significantly decreased by the 8R10Q peptidein both areas. Corresponding Western blot analysis revealed that theRIPA-insoluble or aggregate fraction was decreased by the 8R10Q peptideas compared with PBS. The rescue effect with respect to the neuronalloss and aggregation was accompanied by a significant decrease in thenumbers of GFAP-positive astrocytes (FIG. 8, panels C and D) and that ofIba-l-positive microglia (FIG. 8, panels E and F) in the cortex of the8R10Q-treated R6/2 mice. Furthermore, the R6/2 mice had weak tau signalscompared with the WT mice, but 8R10Q treatment significantly enhancedthis in both cortex and striatum, indicating restoration of the axonalprocesses. Taken together, these results showed that the 8R10Q peptideeffectively decreased the aggregated species of mHtt, and prevented theneurotoxicity induced by the mHtt.

Discussion

In this study, bipartite therapeutic peptides like 8R10Q were shown tobind to mHtt aggregates, decrease mHtt-mediated toxicity, and delay theonset and progress of neurologic dysfunction in HD models. In addition,the therapeutic effect of 8R10Q was observed in a mouse cell linetransiently expressing mHtt, R6/2 transgenic mice, and human HDiPS-derived neurons; its therapeutic effect was independent of theanimal species or length of polyQ in mHtt.

In the bipartite peptide design, the charged sequence played anessential role in preventing the therapeutic peptide itself or misfoldedtarget/peptide hybrid from aggregating. To identify peptides that maydecrease mHtt toxicity, studies using various approaches, such as phagedisplay (Kawasaki et al., Biosci, Biotech, and Biochem (2012) 76:762-6;Lecerf et al., Proc Natl Acad Sci USA (2001) 98:4764-9; Nagai et al.,Hum Mol Genet (2003) 12:1253-9; Magai et al., J Biol Chem (2000)275:10437-42) or other screening methods (Arribat et al., PLoS One(2013) 8:e68775; Lakhani et al., PLoS Computational Biol (2010)6:e1000772; Skogen et al., BMC Neurosci (2006) 7:65) and sequencemodifications (Lanning et al., Biochem (2010) 49:7108-18; Kazantsev etal., Nat Genet (2002) 30:367-76) have previously been conducted.Compared with those approaches, inclusion of the repulsion force by thecharged sequence into peptide design provides several advantages. First,previous studies were mainly target- or disease-specific. In contrast,the present study renders designing therapeutic peptides against variousneurodegenerative diseases possible by choosing a partner sequence witha specific affinity for the misfolded protein/derivative of thedisease-of-interest. This possibility is supported by a separate study,which showed that therapeutic peptides designed by the same principleyielded a similar beneficial outcome in APP/PS1 transgenic mice, awidely used mouse model for AD. Second, it may significantly simplifythe work spent in searching for or in modifying candidate sequences forthe therapeutic peptides. Previous approaches required one to findpeptides of dual functions that would both bind to the misfoldedprotein/derivative, and at the same time, stop the latter fromaggregation, which might require a considerable amount of effort. Also,these efforts would likely need to be reinstated for a differentdisease. In the present design, the affinity sequences could be directlytaken from the misfolded protein/derivative of the particulardisease-of-interest, as already demonstrated in both of the studies.Thus, much work could be eliminated. Furthermore, the strategy may beapplied to the peptides identified in the previous studies to furtherenhance their therapeutic effects.

Example 2 Exemplary Bipartite Therapeutic Peptides for Use in DelayingDisease Onset in APP/PS1 Transgenic Mice

Adult neurodegenerative diseases (NDs) comprise a heterogeneous group ofneurological disorders characterized by disease-specific inclusionbodies (IBs) formed by misfolded peptides/proteins. Alzheimer's disease(AD) is the most common ND, with signature IBs, amyloid plaques, andneurofibrillary tangles. An imbalance in the production and clearance ofmisfolded amyloid β (Aβ) peptide and its variants is considered theprimary cause for the pathogenesis of AD. A modular peptidic designcombining polyarginines (PolyR) and a peptide derived from thepathogenic peptide/protein forming IBs is discussed below. The designedbipartite peptides were expected to have target-specific affinity and tobe able to prevent misfolded peptide/protein self-aggregation by thecharge repulsion conferred by PolyR. A designed bipartite peptideR₈-Aβ(25-35) and its D form derivative ^(D)R₈-Aβ(25-35) were found toprevent Aβ₄₀ from forming amyloid fibrils by circular dichroism andelectron microscopy. When mixed at a 1:1 ratio, both peptidessignificantly decreased the toxicity of Aβ₄₀ to the Neuro2a cells. Dailyintranasal administration of PEI-conjugated R₈-Aβ(25-35) peptide from 4months of age significantly ameliorated the memory deficits of the 8month-old APPxPS1 double transgenic mice compared with PEI-treatedcontrol group as demonstrated by a Morris water maze test. The level oftotal Aβ₄₀ and Aβ₄₂ in the cortex and hippocampus were remarkablyreduced; also, the number of amyloid plaques were consistently reduced.Taken together, the modular design combining polyR with a peptide fromthe pathogenic peptide/protein, like R₈-Aβ(25-35), produced desirabletherapeutic effects and could be easily adopted to design therapeuticpeptides for other diseases characterized by IBs.

Methods and Materials Peptide Synthesis

The peptides were prepared by the batch fluorenylmethoxycarbonyl(fmoc)-polyamide method. Chen et al., Protein Sci (2001) 10:1794-1800.The sequence of Aβ 40: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ IDNO: 20). The sequence of R8A β 25-35: RRRRRRRRGSNKGAIIGLM (SEQ ID NO:3); ^(D)R8A β 25-35 had the same sequence as R8A β 25-35, but the L-formarginines were replaced by D-form arginines. The C-terminal carboxylgroup was amidated using Rink Amide AM resin (Novabiochem, Billerica,Mass., USA) as the solid support. Fmoc-amino-acid derivatives (4equivalents) (Anaspec, Freemont, Calif., USA) were coupled on the resin(1 equivalent) using benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (4 equivalents) and 4.45% (v/v) N-methyl morpholinein dimethylformamide (DMF). The Fmoc cleavage step was performed using20% piperidine in DMF. Side-chain deprotection and peptide cleavage fromthe resin were performed simultaneously by stirring the resin with amixture of 9.4 ml of trifluoroacetic acid, 0.25 ml of water, 0.25 ml ofethanedithiol, and 0.1 ml of triisopropylsilane at room temperature for1-2 hours, then the resin was removed by passing the reaction mixturethrough a G2 glass funnel. The crude peptide was precipitated from thefiltrate by the addition of three volumes of ice-cold methyl t-butylether (MTBE) and centrifugation at 2000 g for 15 minutes at 4° C., thenwashed twice with MTBE, and dried under a vacuum. The precipitatedpeptide was purified by reverse-phase HPLC using a Vydac C18 column (10mm×250mm) and acetonitrile-water mixtures containing 0.1%trifluoroacetic acid. Peaks were analyzed on a matrix-assisted laserdesorption ionization (MALDI) mass spectrometer and those containing thedesired product were lyophilized and stored at −20° C. To synthesize thePEI-conjugated peptide R₈-A β (25-35)-PEI, PEI was conjugated to theC-terminal carboxyl group of the peptide. Fmoc-Met-Wang resin (Anaspec)was used as the solid support during synthesis. To avoid interferencewith PEI conjugation by the N-terminal amino group, the N-terminal groupof the peptide was acetylated using 4 equivalents of acetic anhydrideinstead of an amino acid derivative in the final synthetic step.

Circular Dichroism (CD) Spectroscopy

The peptide samples were dissolved in 75% trifluoroethanol as 1.4 mMstock solutions, then diluted into 20 mM sodium phosphate buffer with150 mM KCl (pH 7) to a final peptide concentration of 30 μM andincubated at 25° C. After the indicated times, the samples were placedin a 1-mm cell and the CD spectra between 200 and 250 nm were recordedon a J-715 CD spectrometer (JASCO, Japan). The band width was set to 2nm and the step resolution was 0.05 nm. Each sample was scanned twiceand the recorded spectra were averaged to get the final spectrum.

Transmission Electron Microscopy

The samples were deposited on carbon-coated 300-mesh copper grids,incubated for 3 min for absorption, and then washed by water. Negativestaining was carried out by staining with 2% uranyl acetate for 1.5 min.After air drying, the samples were viewed using a Hitachi H-7000electron microscope (Hitachi, Tokyo, Japan).

Cell Viability Assay

Mouse N2a neuroblastoma cells (ATCC) were cultured in Dulbecco'smodified Eagle's medium (DMEM) (HyClone, USA) supplemented with 10%fetal bovine serum (FBS; HyClone, USA) in 5% CO₂ at 37 ° C. For the cellviability assay, the cells were harvested, suspended at a density of350,000 cells/mL in DMEM, and 100 μL of each sample was plated in eachwell of a 96-well CellBIND polystyrene microplate (Corning, USA).Because the cytotoxicity experiments lasted for up to 4 days, cellproliferation was blocked using a medium without FBS. The plates werethen incubated at 37° C. under 5% CO₂ for 24 h to allow the cells toattach to the well. The peptides were dissolved in DMSO as 6 mM stocksolutions. Five microliters of the stock solution (6 mM) was dilutedwith 95 μL of PBS (20 mM sodium phosphate buffer, 150 mM KCl, pH 7.0),and then the sample was immediately added to 900 μL of fresh DMEM mediumto give a peptide concentration of 30 μM. To test the efficacy of thepeptide inhibitor, equal volumes of Aβ40 and peptide inhibitor stocksolutions were pre-mixed and then diluted into PBS to make a finalconcentration of 30 μM for each peptide. The diluted peptide solutionswere pre-incubated for 24 h at room temperature with shaking (50 rpm)before being added to the cultures. The medium in the well of the96-well plate was replaced with 100 μL of peptide-containing medium andthe plate was incubated for 48 h. Cell viability was determined usingthe MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)toxicity assay. Shearman et al., J Neurochem (1995) 65:218-27. Tenmicroliters of 5 mg/mL of MTT in PBS was added to each well, and then,after incubation for 4 h, the medium was removed and the MTT crystalswere dissolved in 100 μL of 90% isopropanol, 0.5% SDS, and 40 mM HCl,and their absorption at 570 nm was measured. Cell viability wascalculated by dividing the absorbance of the wells containing peptidesamples by that of the wells without any added peptide. The experimentwas repeated at least four times and eight replicate wells were used foreach sample and control in each independent experiment.

Synthesis of PEI-Conjugated Peptide

Three milligrams of acetylated R₈-A β (25-35) dissolved in 2.5 mLdimethyl sulfoxide (DMSO) was slowly mixed with 150 μL1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (600 mM in 0.1 MMES, 0.5 M NaCl, pH 6) and with 150 μL N-hydroxysuccinimide (NHS) (1200mM in 0.1 M MES, 0.5 M NaCl, pH 6) subsequently. The reaction mixturewas reacted at room temperature for 30 min with gentle shaking (70 rpm).To the mixture, 180 μL polyethylenimine (PEI) was added and reacted atroom temperature overnight with gentle shaking (70 rpm). ThePEI-conjugated peptide, named R₈-A β (25-35)-PEI, was separated fromunreacted PEI and R₈-A β (25-35) by reverse-phase HPLC using a Vydac C18column (10 mm×250 mm) and acetonitrile-water mixtures containing 0.1%trifluoroacetic acid. Peaks were analyzed on a matrix-assisted laserdesorption ionization (MALDI) mass spectrometer and those containing thedesired product were lyophilized and stored at −20 ° C.

Intranasal Administration

APP×PS1 transgenic mice (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax , purchasedfrom Jackson Laboratories (USA), were bred and genotyped following thevendor's protocols. Borchelt et al., Neuron (1996) 17:1005-13; andJankowsky et al., Biomol Eng (2001) 17:157-65. The mice had access tofood and water ad libitum and were kept on a 12:12 h light-dark cycle.PEI and R8A β 25-35-PEI were dissolved in 100 mM NaH₂PO₄/138 mM KCl (pH5) to a final concentration of 400 μM. For intranasal administration,2.5 μL PEI or R₈-A β (25-35)-PEI was given to each nostril of one mousewhen they were 3 months of age for six days/week until they were 8months of age. Mice were then tested with a Morris water maze. Treatmentwere resumed after a 6-week break for the water maze test, and continueduntil the mice were 12 months of age.

ELISA Assays for Total Aβ40 and Aβ42

The levels of Aβ40 and Aβ42 in mouse brain homogenate were detectedusing ELISA kits (Invitrogen, Md., USA) according to the manufacturer'sinstructions. Briefly, the cortical or hippocampal tissue was weighedand homogenized at 4° C. in the cell extraction buffer provided in thekit, supplemented with protease inhibitor cocktail (Sigma, St. Louis,USA). The homogenates were then centrifuged in Eppendorf tubes at 13000rpm at 4 ° C. for 10 min and the concentration of proteins in thesupernatant was measured using the microBCA protein assay (Thermo, Ill.,USA). The APP levels were adjusted in accordance with the proteinlevels.

ELISA Assays for Insoluble Aβ40 and Aβ42

Half of the frozen cortical and hippocampal tissue samples werehomogenized in 1 mL tapered tissue grinders in 400 μL of ice-cold TBScontaining a protease inhibitor cocktail (P8340, Sigma). The homogenateswere then transferred to 1.5 mL Eppendorf tubes and centrifuged at 20000g for 20 min at 4° C. The supernatant contained soluble Aβ, whereas theTBS-insoluble pellet contained insoluble Aβ proteins. The TBS-insolublepellet was suspended in 70% formic acid, sonicated for 1 min, and thencentrifuged at 20000 g for 20 min at 4° C. The final supernatantcontaining the solubilized Aβ was removed and neutralized with 20volumes of 1 M Tris base. The protein concentrations of the samplescontaining the solubilized “insoluble” Aβ was quantified using theBradford protein assay (Bio-Rad #500-0006).

Morris Water Maze Task

The maze was made of white opaque plastic with a diameter of 120 cm andcontained 40 cm high walls. It was filled with milk/water at 25 ° C. Asmall escape platform (10×6.5×21.5 cm) was placed at a fixed position inthe center of one quadrant, 25 cm from the perimeter, and was hidden 1cm beneath the water's surface. The room contained a number of fixedvisual cues on the walls. The acquisition trial phase consisted of 5training days (Days 1-5) and four trials per day with a 15 mininter-trial interval. Four points equally spaced along the circumferenceof the pool (North, South, East, and West) served as the startingpositions, which were randomized across the four trials daily. If ananimal did not reach the platform within 90 s, it was guided to theplatform, where it stayed for 15 s. The path length and escape latencieswere recorded (n=10 per group). Acquisition data, such as time taken toreach the escape platform and path length were analyzed by two-wayrepeated measures ANOVA. On Day 5, after finishing three trials, a probetrial was performed in order to assess the mouse's spatial memory. Theplatform was removed from the maze and animals were allowed to swimfreely for 90 s and the swimming path was recorded and analyzed.

Cytometric Bead Array

A cytometric bead array (CBA, mouse inflammation kit; BD Biosciences)was used to quantitatively measure cytokine expression levels in thecontrol and treated mouse brain tissue lysates. CBA was also performedto measure the cytokines from different brain regions (cortex andhippocampus). This method quantifies soluble particles, in this case,cytokines, using a fluorescence-based detection mechanism. The beads,coated with the desired cytokine, IL-6 and IL-1β, reacted with the testlysates and standards, to which fluorescence dyes were then added. Theassay was performed according to the manufacturer's instructions andanalyzed on the FACS Calibur (Becton Dickinson). Analysis was performedusing CBA software that allows the calculation of cytokineconcentrations in unknown samples. Soldan et al., J Neuroimmunol (2004)146:209-15.

Thioflavin S Staining

The mice were perfused with ice-cold PBS (136.89 mM NaCl, 2.68 mM KCl,1.62 mM KH₂PO4, and 10.14 mM Na₂HPO₄, pH 7.4) buffer and 4%paraformaldehyde (PFA)/PBS. Brains of the perfused mice were thenpost-fixed in 4% PFA/PBS with gentle shaking in a cold room for another24 hours. Post-fixation, the brain samples were washed with PBS bufferand preserved in a 70% ethanol solution. PFA-fixed brain tissues weredehydrated by a semi-enclosed benchtop tissue processor (Leica TP1020).Paraffin blocks containing the dehydrated brain samples were prepared bya Leica EG1150 H. Coronal sections with 5 μm thickness were cut on amicrotome (Leica RM2235). Two brain sections were transferred with abrush, put onto the surface of a water bath, floated onto the surface ofclean glass slides, and placed on a 34° C. warming block for severalhours. Paraffin slides were deparaffinized and rehydrated with xylene,absolute ethanol, 95% ethanol, 70% ethanol, and water, sequentially.Rehydrated slides were given 1% (w/v) thioflavin S (ThS) solution for 10min at room temperature while protected from light. The slides werewashed with 80% ethanol and water to remove excess ThS and to facilitatevisualization. ThS-positive signals were then visualized with afluorescence microscope equipped for evaluation of green fluorescence,and the plague number, plague area, and plague size were analyzed byImageJ.

Radiosynthesis of [¹¹C] PIB

The radiosynthesis of [¹¹C] PIB was performed by using [¹¹C]methyltriflate according to the method described previously with minimalmodification. Takalo et al., Am. J. Neurodeger. Dis. (2013), 2:1-14.Briefly, [¹¹C] methyl bromide was produced by the multi-pass brominationof [¹¹C] methane. Subsequently, [¹¹C] methyl bromide was eluted from atrap and converted to [¹¹C] methyltriflate by passing through apreheated silver triflate column. [¹¹C] methyltriflate was carried by ahelium stream (20 ml/min) into 350 μl of anhydrous methylethylketonecontaining 1.5 mg of 2-(4′-aminophenyl)-6-hydroxybenzothiazole. Afterthe trapping was finished, the reaction mixture was heated at 75° C. for2 min and then 0.4 ml of the HPLC mobile phase was added to the reactionmixture for HPLC purification. HPLC purification was performed on aWaters Bondapak column (10 m, 7.8 mm ID×300 mm) using a mobile phase ofacetonitrile/0.01 M H₃PO₄ (40/60) at a flow rate of 5.0 ml/min. Theradioactive fraction corresponding to [¹¹C] PIB was collected in abottle containing 30 ml of pure water and passed through a C18 Sep-PakPlus cartridge, then washed with 10 ml of pure water, eluted with 1 mlof ethanol and 10 ml of sterile normal saline, and passed through a 0.22μm sterile filter for quality analysis and animal experiments.Radiochemical purity was greater than 99%, as determined by analyticalHPLC. The specific activity was 152±52 GBq/μmol at the end of thesynthesis.

In vivo Small-Animal Positron Emission Tomography Imaging

All PET scans were performed using Triumph pre-clinical tri-modality(LabPET/X-SPECT/X-O CT) imaging system (TriFoil Imaging, USA), whichprovides 31 transaxial slices 1.175 mm (center-to-center) apart, a 100mm transaxial FOV, and a 37 mm axial FOV for the LabPET sub-system. Thedigital APD detector technology delivers high spatial resolution betterthan 1 mm and a high recovery coefficient. Before the scans, all of themice were kept warm with a heating lamp. After induction with 2.0%isoflurane, the mice were placed with their heads in the center of thefield of view and were fixed in the prone position. A 20 min static dataacquisition was performed in the 3D list mode with an energy window of350-650 keV at 20 min following a [¹¹C] PIB (36.7±2.6 MBq; volume <0.25ml) injection via the tail vein. The emission data were normalized andcorrected for the tracer decay time. All list mode data were sorted into3D sinograms, which were then single-slice Fourier rebinned into 2Dsinograms. Summation images from 20 to 40 min after the [¹¹C] PIBinjection was reconstructed using a MLEM algorithm, and the resultingimage volume consisted of 240×240×31 voxels, with voxel size of0.25×0.25×1,175 mm³.

PET Data Analysis

All imaging data were processed and analyzed with PMOD 3.5 softwarepackage (Pmod Technologies, Ziirich, Switzerland). The PET image datasetwere converted to an absolute measure of radioactivity concentration(kBq/cc) using a phantom-derived calibration factor before beingnormalized to the injected dose (ID) of [¹¹C] PIB and the body mass ofthe animal. This normalization enabled the comparison of the brainradioactivity concentration of animals of different weights. Static PETimages were co-registered with a mouse T2-weighted MRI brain atlas basedon PMOD as an anatomic reference. Image origins were set to Bregma (0,0) according to the MRI atlas and the atlas was used for VOI definition.[¹¹C] PIB uptake was evaluated in the 4 regions of interest, namely: thecortex, the hippocampus, the amygdala, and the olfactory bulb.Standardized uptake values (SUV) were obtained for each VOI by dividingthe mean [¹¹C] PIB activity by the injection dose and the body weight(gram in grams). Thereafter, regional [¹¹C] PIB uptake in the targetregion was normalized by [¹¹C] PIB uptake in the cerebellum which wastaken as the reference region (ratio to cerebellum). Manook et al., PLoSOne (2012) 7:e31310; Poisnel et al., Neurobiol Aging (2012) 33:2561-71.

Results Affinity Plus Charge Modular Design for Therapeutic Peptides

A rational design to synthesize therapeutic peptides that might reducetoxic misfolded protein/derivative across various neurodegenerativediseases was proposed. The design was built on a principle of modularassembly of sequences with affinity and charge. The conjectured peptidewas composed of an affinity sequence derived from the self-aggregatingregion of the misfolded protein/derivative flanked on one side or bothsides by a stretch of charged amino acids. The rationale behind thisdesign was that the affinity sequence would facilitate the binding oftherapeutic peptides to the misfolded protein/derivative because of itspropensity to aggregate; the charged sequence would then prevent thetherapeutic peptide itself and the misfolded protein/peptide hybrid fromaggregation by the repulsion force of charges (FIG. 1). Theoretically,an amino acid with either a positive or negative charge could achievethe goal. In the present design, positively charged arginine was chosenbecause polyarginine (poly-Arg) had been shown to facilitate thepenetration of therapeutic peptides into the cytoplasmic and nuclearcompartments of target cells or organs. Mitchell et al., J Pept Res(2000) 56:318-25.

Conformational Study of Aβ₄₀, R₈-Aβ(25-35), and ^(D)R₈-Aβ(25-35)

Polyethylenimine (PEI) cationized proteins can transduce across cellmembranes. Futami et al., J Biosci Bioeng (2005) 99:95-103; Futami etal., Expert Opin Drug Discov (2007) 2:261-9; Kitazoe et al., J Biochem(2005) 137:693-701; Kitazoe et al., Biotechnol J (2010) 5:385-92; Murataet al., J Biochem (2008) 144:447-55; Murata et al., J Biosci Bioeng(2008) 105:34-8. PEI is protease resistant and has higher chargerdensity than a poly-Arg or poly-lysines fragment. PEI-conjugated greenfluorescent protein could pass through the blood-brain barrier, andenter into brain by intranasal administration. Loftus et al., Neurosci(2006) 139:1061-7.

To investigate the biophysical property of these peptides concerningamyloid formation, Aβ₄₀, R₈-Aβ(25-35), and ^(D)R₈-Aβ(25-35) dissolved in20 mM sodium phosphate buffer with 150 mM KCl (pH 7) were individuallyincubated at 25° C. The circular dichroism (CD) spectra were recorded atvarious time points as indicated. As expected, the intensity of thenegative ellipticity at 218 nm of Aβ₄₀ spectrum increased with time(FIG. 10, panel A), consistent with its known ability to form amyloidfibrils as shown by the transmission electron microscopy (TEM) (FIG. 10,panel D). In contrast, the CD spectra of R₈-Aβ(25-35) showed negativeellipticity at 200 nm, indicative of random coil structure (FIG. 10,panel B). Similarly, the ^(D)R₈-Aβ(25-35) also had CD spectra consistentwith random coil structure, which lacked strong negative ellipticity at200 nm due to the presence of eight D-form arginines in the peptide(FIG. 10, panel C). The CD spectra of both peptides remained largelyunchanged with the incubation time. The TEM showed that R₈-Aβ(25-35) and^(D)R₈-Aβ(25-35) formed amorphous aggregates in the same condition (FIG.10, panels E and F). No amyloid fibrils were observed.

To examine whether R₈-Aβ(25-35) or ^(D)R₈-Aβ(25-35) could interfere withthe amyloidogenesis of Aβ₄₀, the CD spectra of Aβ₄₀ mixed withR₈-Aβ(25-35) or ^(D)R₈-Aβ(25-35) at a 1:1 ratio were measured. As shownin FIG. 10, panels G and H, the signal at 218 nm is hardly changed whenR₈-Aβ(25-35) or ^(D)R₈-Aβ(25-35) co-incubated with Aβ₄₀. The kinetictraces of amyloidogenesis in FIG. 10, panel I suggested that theexistence of R₈-Aβ(25-35) or ^(D)R₈-Aβ(25-35) largely increase the lagtime. After long time incubation, the TEM images of the peptide mixturesshowed hybrids of fibrils and amorphous aggregates (FIG. 10, panels Jand K). These results showed that R₈-Aβ(25-35) and ^(D)R₈-Aβ(25-35)could interact with Aβ₄₀ and interfere with its self-aggregation, hencesignificantly delay or decrease the formation of Aβ₄₀ amyloid fibrils.

Attenuation of Aβ₄₀ cytotoxicity by R₈-Aβ(25-35) and ^(D)R₈-Aβ(25-35)

To test whether R₈-Aβ(25-35) or ^(D)R₈-Aβ(25-35) could attenuate thecytotoxicity of A β₄₀, the MTT assays were performed to measure theviability of Neuro2a cells (a mouse neuroblastoma cell line) treatedwith peptides as indicated. Aβ₄₀ (μM) exerted significant cytotoxicityand only 30% of cells survived. By contrast, both R₈-Aβ(25-35) and^(D)R₈-Aβ(25-35) had no detectable toxicity to the N2a cells (FIG. 11,panel A). Both R₈-Aβ(25-35) and ^(D)R₈-Aβ(25-35) decreased Aβ₄₀ toxicityand increased the cell viability from 30% to 70-75%. For comparison,when Aβ40 was mixed with Aβ (25-35), no significant change in cellviability was observed. The data showed that both R₈-Aβ(25-35) and^(D)R₈-Aβ(25-35) peptides had therapeutic potential in amyloid-inducedtoxicity.

Therapeutic Effect of R₈-Aβ(25-35) in APP/PS1 Transgenic Mice

To test the effect of R₈-Aβ(25-35) to prevent the deterioration ofmemory in vivo, PEI or PEI-conjugated R₈-Aβ(25-35) was givenintranasally to wild-type or APP/PS1 transgenic mice starting from 3-4months of age. The water maze assay was performed when the mice reached8 months of age. As shown in FIG. 12, panel A, the wild-type micetreated with PEI and R₈-Aβ(25-35)-PEI showed no clear difference in thelearning curve of finding the hidden platform. The R₈-Aβ(25-35)-treatedAPP/PS1 mice, on the other hand, exhibited significantly faster learningthan transgenic mice treated with PEI. In addition, R₈-Aβ(25-35)peptide-treated APP/PS1 mice performed better at the probe testevidenced by more crossings of (FIG. 12, panel C) and longer time spentin the quadrant where the probe used to be than those treated with PEI(FIG. 12, panel B).

The change in the level of Aβ peptide was next examined via ELISA. Asshown in FIG. 13, at 8 months of age, the level of Aβ₄₀ and Aβ₄₂decreased by 86% and 30%, respectively in the cortex of R₈-Aβ(25-35)peptide-treated APP/PS1 mice as compared with that of PEI-treatedtransgenic mice. Similarly, the levels of Aβ₄₀ and Aβ₄₂ decreased by 73%and 60%, respectively in the hippocampus of the former mice comparedwith the latter.

Consistently, histological sections stained with Thioflavin Schemifluorescent dye revealed deposition of multiple green-fluorescentamyloid plaques in both cortex and hippocampus of the 8 month-oldPEI-treated APP×PS1 mice, which was significantly reduced in theage-matched peptide-treated APP×PS1 mice (FIG. 14, panel A). As shown inFIG. 14, panel B, the number of plaques was significantly reduced in thecortex and hippocampus of the peptide-treated mice compared with that inthe corresponding regions of the PEI-treated mice. The total area ofamyloid plaques was correspondingly decreased in the both cortex andhippocampus of the peptide-treated mice vs. that in the PEI-treatedcontrols (FIG. 14, panel C). To further eliminate the effect that mightcontribute to the positive results by the variation in the size ofindividual brain sections, the percentage of the area of the cortex andhippocampus that harbored amyloid plaques was calculated; the peptidetreatment again significantly decreased the percentage of the plaqueareas of both regions in comparison with the PEI control treatment (FIG.14, panel D). Whether the size of individual plaques were altered bytreatment was next examined; as shown in FIG. 14, panel E. Although thepeptide treatment decreased the average size of individual plaques, thedifference failed to reach statistical significance. The data indicatedthat the peptide treatment effectively slowed down the accumulation ofamyloid plaques and the clinical impairment of the memory. Amyloiddeposition induced neuroinflammation which contributed importantly tothe diseases in these mice. R₈-Aβ(25-35)-PEI effectively decreased thelevel of pro-inflammatory cytokines interleukin (IL)-6 and IL-1β in thecortex (FIG. 15).

Therapeutic Effect of Peptide after a Suspension for 4 Weeks

During the water maze tests, the treatment was adjourned for about 4weeks. To examine whether the therapeutic effect could be maintained orresumed after a suspension of treatment, administration of PEI orpeptide was resumed and continued until these mice reached 12 months ofage. The burden of amyloid plaques was quantified with microPET usingthe tracer Pittsburg compound B (PiB). As shown in FIG. 16, panels A andB, the R₈-Aβ(25-35) peptide-treated APP×PS1 mice had a much lower signalin the cortex, hippocampus, amygdala and olfactory bulb as compared withthe PEI-treated mice, which is consistent with a beneficial therapeuticeffect at this age. ELISA analyses revealed a decrease in total Aβ₄₀ by16% and 21% in the cortex and hippocampus, respectively (FIG. 17, panelsA and D). No statistical difference was detected in the level of totalAβ₄₂. Consistent with the results of microPET (17-35% reduction), theinsoluble pools of Aβ₄₀ and Aβ₄₂ were both decreased by 25-30% in thecortex or hippocampus of the peptide-treated APP XPS/mice compared withthose in PEI-treated mice (FIG. 17, panels C and D).

Discussion

In this study, it was demonstrated that the peptide R₈-Aβ(25-35) or itsD-form counterpart could reduce the formation of amyloid fibrils by Aβ₄₀peptide in vitro, and amyloid plaques and disease manifestation in vivo.Intranasal administration of R₈-Aβ(25-35) peptide also exhibitedbeneficial therapeutic effect in APP/PS1 transgenic mice. Thus, the dataindicates that therapeutic peptides designed based on theaffinity/charge modular principle described herein would be expected tobe therapeutically effective in treating neurodegenerative diseases,particularly those that involve abnormal protein aggregation.

In the present design, the charged sequence not only enhanced cellpermeability and bioavailability of the bipartite peptides, but also hadan essential role in the peptidic therapy by providing a repulsion forceto prevent the misfolded target from further aggregation. The chargemoieties were introduced at both N- and C-ends of the peptide. TheN-terminal positive charges were introduced by the guanido groups in theside-chains of poly-Arg; while the C-terminal positive charges, by thechemical modification of C-terminal carboxyl group withpolyethylenimine. In addition, in comparison with another Aβaggregation-inhibiting PEI-coupled V24P(10-40) peptide, R₈-Aβ(25-35)performed better.

Although many therapeutic peptides have been designed, only few of themwere tested in vivo (Shukla et al., FASEB J (2013) 27:174-86;Frydman-Marom et al., Angew Chem Int Ed (2009) 48:1981-6; Funke et al.,ACS Chem Neurosci (2010) 1:639-48; Permanne et al., FASEB J (2002)16:860-2; van Groen et al., ChemMedChem (2008) 3:1848-52). In thisstudy, the feasibility of the administration of therapeutic peptidicprodrugs through an intranasal route was demonstrated. When combinedwith technology in delivery, the study showed a proof of a therapeuticprinciple for neurodegenerative diseases through intranasal delivery.The dose used in this study was estimated to be 2 nmoles per day, whichwas quite low compared with previous studies. Frydman-Marom et al.,Angew Chem Int Ed (2009) 48:1981-6; Funke et al., ACS Chem Neurosci(2010) 1:639-48; Permanne et al., FASEB J (2002) 16:860-2; van Groen etal., ChemMedChem (2008) 3:1848-52.

Given the fact that amyloid plaques form extraordinarily rapidly invivo, and might even do so as fast as in 1-2 days (Meyer-Luehmann etal., Nature (2008) 451:720-4), it was likely that after a 4-weekinterruption of treatment, deposition of amyloid plaques in theR₈-Aβ(25-35)-PEI-treated mice had approached or reached the level inPEI-treated mice. It was encouraging that the resumption of peptidetreatment remained beneficial in 12 month-old mice. These findingsindicate further prevention of amyloid plaques from deposition can beattained, even after a period of interruption. In fact, the preliminarytests for liver and kidney function indicated no clear toxicity in themice receiving the peptides. In sum, intranasal administration of thedesigned bipartite peptides provided an effective and user-friendlypreventative and therapeutic approach to treat aggregation-causedneurodegenerative diseases.

Example 3 Intranasal Delivery of a Bipartite Peptide Reduced AmyloidBurden in the Brains of APP/PS1 Transgenic Mice

A “scavenger peptide”, V24P(10-40), designed to decrease Aβ accumulationin the brain, was conjugated to polyethylenimine (PEI), and tested as apreventive and/or therapeutic strategy for Alzheimer's disease (AD) inthis study. This PEI-conjugated V24P(10-40) peptide was deliveredintranasally to the APP/PS1 double transgenic mice of 4 months of age asnasal drops for four months. Compared with control values, peptidetreatment reduced the amount of Aβ peptides by 72% of Aβ40 and 40% ofAβ42 in the hippocampus, and by 87% of Aβ40 and 32% of Aβ42 in thecortex. After treatment for 8 months, amyloid load, as quantified byPittsburg compound B microPET imaging, was decreased significantly inthe hippocampus, cortex, amygdala, and olfactory bulb. The datademonstrate that the intranasally delivered scavenger peptide iseffective in decreasing Aβ plaque formation in the brain. Nasalapplication of peptide drops is user-friendly, and could be furtherdeveloped for preventative and therapeutic purposes with respect to ADand other neurodegenerative diseases, including those described herein.

Materials and Methods Synthesis of the PEI-Conjugated Peptide

All peptides were synthesized by the batch fluorenylmethoxycarbonyl(fmoc)-polyamide method. Chen et al., Protein Sci (2001) 10:1794-1800.To generate PEI-conjugated V24P(10-40), PEI was conjugated to theC-terminal carboxyl group of the peptide, and the N-terminal group ofV24P(10-40) was acetylated to avoid dimerization or cyclization of thepeptide during PEI-conjugation reaction and to provide protectionagainst exopeptidases. Acetylated V24P(10-40) (4.8 mg) dissolved in 4 mLof dimethyl sulfoxide was slowly mixed with 240 μL of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (600 mM in 0.1 M MES, 0.5M NaCl, pH 6), then 240 μL of N-hydroxysuccinimide (1200 mM in 0.1 MMES, 0.5 M NaCl, pH 6) was added, and the mixture was reacted at roomtemperature for 30 min with gentle shaking (70 rpm). PEI (288 μL) wasadded and the mixture was incubated overnight at room temperature withgentle shaking (70 rpm). The PEI-conjugated peptide, V24P(10-40)-PEI,was separated from unreacted PEI and V24P(10-40) by reverse-phase HPLC.

Animal Experiment

APP/PS1 transgenic mice (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax), purchasedfrom Jackson Laboratories (USA), were bred and genotyped as described onthe Jackson website. Jankowsky et al., Biomol Eng (2001) 17:157-65;Borchelt et al., Neuron (1996) 17:1005-13. The mice had access to foodand water ad libitum and were kept on a 12:12 h light-dark cycle. PEIand V24P(10-40)-PEI were dissolved at a concentration of 400 μM in 100mM NaH₂PO₄, 138 mM KCl, pH 5, and 4-month-old mice was given 2.5 μL ofPEI (as the control) or V24P(10-40)-PEI in each nostril six days perweek for the indicated period.

ELISA Assays for Aβ40 and Aβ42

Concentrations of Aβ40 and Aβ42 in mouse brain homogenate were measuredusing ELISA kits (Invitrogen, Md., USA) according to the manufacturer'sinstructions. Briefly, the cortical or hippocampal tissue fromPEI-treated or V24P(10-40)-PEI-treated APP/PS1 mice was weighed andhomogenized at 4 ° C. in the cell extraction buffer provided in the kit,supplemented with protease inhibitor cocktail (Sigma, St. Louis, USA),then the homogenates in Eppendorf tubes were centrifuged at 13000 rpm at4 ° C. for 10 min and the concentration of protein in the supernatantwas measured using the microBCA protein assay (Thermo, Ill., USA). Toperform the ELISA, the supernatants were diluted 10-fold and the Aβ40and Aβ42 concentrations were normalized to the protein concentration andexpressed as ng/mg of protein.

In vivo MicroPET

[¹¹C] PIB was generated using ^([11C]) methyltriflate using a previouslydescribed method with minimal modification. Manook et al., PLoS One(2012) 7:e31310. PET scans were performed using a Triumph pre-clinicaltri-modality (LabPET/X-SPECT/X-O CT) imaging system (TriFoil Imaging,USA). The mice were kept warm with a heating lamp before scanning. Afterinduction with 2.0% isoflurane, the mice were placed with their heads inthe center of the field of view, fixed in the prone position, and thenfreshly synthesized [¹¹C]PiB (36.7±2.6 MBq; volume <0.25 mL) wasinjected via the tail vein. After 20 min, static data acquisition wasperformed for 20 min in 3D list mode with an energy window of 350-650keV. The emission data were normalized and corrected for the tracerdecay time. All list mode data were sorted into 3D sinograms, which werethen single-slice Fourier rebinned into 2D sinograms. Summation imagesfrom 20 to 40 min after [¹¹C]PiB injection were reconstructed using aMLEM algorithm.

All imaging data were processed and analyzed using the PMOD 3.5 softwarepackage (Pmod Technologies, Zurich, Switzerland). The PET image datasetwas converted to an absolute measure of radioactivity concentration(kBq/cc) using a phantom-derived calibration factor before beingnormalized to the injected dose of [¹¹C]PiB and the body mass of theanimal. Static PET images were co-registered with the mouse T2-weightedMRI brain atlas based on PMOD as anatomic reference. Image origins wereset to bregma (0, 0) according to the MRI atlas, which was also used forVOI definition. [¹¹C]PiB uptake in the cortex, hippocampus, amygdala,and olfactory bulb was evaluated. Standardized uptake values wereobtained for each VOI by dividing the mean [¹¹C]PiB activity by theinjection dose and the body weight (in grams). Thereafter, the regional[¹¹C]PiB uptake in the target region was normalized to [¹¹C]PiB uptakein the cerebellum, taken as the reference region. Manook et al., PLoSOne (2012) 7:e31310; Poisnel et al., Neurobiol Aging (2012) 33:2561-71.

Results

Several mutated Aβ40 peptides with different N- and C-terminaltruncations were designed and synthesized (Table 3). Their structuralproperties were first examined by Circular Dichroism (CD) Spectroscopy,the Thioflavin T (ThT) binding assay, and Transmission ElectronMicroscopy (TEM), and then their effects on the formation of Aβ40fibrils and Aβ40 cytotoxicity using a cell viability assay in mouseneuroblastoma Neuro2a (N2a) cells was performed.

TABLE 3 The synthesized peptides used in this study. DP representsD-proline Peptide Serpence Aβ40DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV V24P(1-28)DAEFRHDSGY EVHHQKLVFF AED ^(D)PGSNK V24P(10-40) Y EVHHQKLVFF AED^(D)PGSNKGA IIGLMVGGVV V24P(13-36) HHQKLVFF AED ^(D)PGSNKGA IIGLMVV24P(16-33) KLVFF AED ^(D)PGSNKGA IIG V24P(19-30) FF AED ^(D)PGSNKGA

The sequences in Table 3, from top to bottom, correspond to SEQ ID NOs:20-25.

C-Terminal-Truncated Peptide V24P(1-28) According to the structuralmodel of the Al3 40 fibrils (Petkova et al., Proc Natl Acad Sci (2002)99:16742-7), K28 is located at the beginning of the second β -strand.Without the C-terminal hydrophobic tail following K28, V24P(1-28) hadincreased hydrophilicity compared with A β 40. V24P(1-28) was dissolvedin 20 mM sodium phosphate buffer, pH 7, containing 150 mM KCl(incubation buffer) and then incubated at 25° C. At different timepoints (Day 0 was the time immediately after dissolution), its CDspectra and fluorescence spectra after ThT binding were recorded. The CDspectrum of the 30 μM V24P(1-28) solution was typical of a random coilstructure and was identical at all tested time points (0-12 days) (FIG.18, panel A, left); consistently, the measurement of fluorescence showedno ThT binding (FIG. 18, panel B, left). When the peptide concentrationwas increased to 60 μM, V24P(1-28) remained as a random structure atdays 0-3, but gradually formed a β-sheet structure (FIG. 18, panel A,right), which bound ThT, as shown by the increased fluorescence (FIG.18, panel B, right). TEM showed that 60 μM V24P(1-28) formed amyloidfibrils (FIG. 18, panel E, left). The fibrils were straight andlaterally associated, in contrast with the twisted morphology commonlyreported for A β 40 fibrils. Chang et al., J Mol Biol (2009)385:1257-65; Meinhardt et al., J Mol Biol (2009) 386:869-77; Goldsburyet al., J Struct Biol (2000) 130:217-31.

N-Terminal-Truncated Peptide V24P(10-40)

The N-terminal region of Aβ 40 is hydrophilic and was reported not to beinvolved in the amyloid cross-β structure. Petkova et al., Proc NatlAcad Sci USA (2002) 99:16742-7. To verify whether this hydrophilicsegment was important in the design of a peptide inhibitor, a V24Pmutant peptide, denoted V24P(10-40), with the first 9 N-terminal aminoacid residues truncated was chemically synthesized and dissolved in theincubation buffer. Unlike 30 μM V24P forming a random coil structure(Chang et al., J Mol Biol (2009) 385:1257-65), the CD spectra of 30 μMV24P(10-40) exhibited a characteristic β -sheet signal evidenced bynegative ellipticity at 218 nm (FIG. 18, panel C, left) and,consistently, fluorescence emission at 487 nm appeared in the ThTbinding assay (FIG. 18, panel D, left). These data showed thatV24P(10-40) was more prone to aggregate than V24P. At the concentrationof 60 μM, more β -aggregates were formed (FIG. 18, panels C and D,right). TEM showed that V24P(10-40) formed amorphous aggregates (FIG.18, panel E, right).

Structural Studies on Shorter Peptide

In order to determine the shortest sequence for self-aggregation, anadditional three peptides with shorter lengths, V24P(13-36),V24P(16-33), and V24P(19-30) were designed, and their CD spectra andfluorescence spectra after binding ThT immediately after dissolution atconcentrations of 30, 60, and 90 μM were examined.

The CD spectra showed that V24P(13-36) formed a random coil structure at30 μM (FIG. 19, panel A, top) and the ThT fluorescence spectra showedthat β-aggregates formed at 90 μM (FIG. 19, panel B, top). V24P(13-36)lacked several hydrophobic residues (Y10,V12,V39,V40) present in theV24P(10-40), and thus required a higher peptide concentration (90 μM) toform β-aggregates compared with V24P (60 μM) or V24P(10-40) (30 μM).

In contrast, the CD spectrum of V24P(16-33) showed a strong peak at 204nm and a trough at 228 nm (FIG. 19, panel A, center panel), a patternindicative of a type I β-turn (Kelly et al., Curr Protein Pept Sci(2000) 1:349-84; Kelly et al., BBA-Protein Proteom (2005) 1751:119-39).The weak fluorescence of 90 μM V24P(16-33) suggested that only a smallamount of β-aggregates formed (FIG. 19, panel B, center).

V24P(19-30) showed a random coil structure. When the peptideconcentration was 60 μMor higher, the CD spectra showed a peak at around220 nm (FIG. 19, panel A, bottom), similar to that of an extended 3₁₀helix or a poly(Pro) II helix (Kelly et al., BBA-Protein Proteom (2005)1751:119-3930). No fluorescence emission at 487 nm was observed at anyconcentration (FIG. 19, panel B, bottom), suggesting that L17, V18, I31,and 132 were responsible for ThT binding to V24P(16-33) aggregates.

Selection of Scavenger Peptide

A single substitution (V24→^(D)P) in Aβ40 dramatically affects thestructural behavior and decreases the toxicity of Aβ 40. (Chang et al.,J Mol Biol (2009) 385:1257-65). FIG. 20, panel A shows that all of the^(D)P-containing peptides tested were much less cytotoxic than Aβ 40.Compared with V24P, V24P(10-40) formed β -aggregates at a lower peptideconcentration, suggesting that it had a higher propensity to aggregateand might have a greater inhibitory effect on Aβ 40 cytotoxicity. In thecytotoxicity assay, Aβ 40 peptide, when mixed with V24P(10-40) at anequi-molar ratio, had the lowest cytotoxicity. FIG. 20, panel B.Therefore, V24P(10-40) was chosen as the scavenger peptide in thefollowing animal studies.

The Inhibitory Effect of V24P(10-40) on Amyloid Formation was Specificfor Aβ40

Many peptide inhibitors were designed based on a short recognitionsequence, such as “KLVFF” (SEQ ID NO: 26), but the target specificity ofthis sequence for different amyloid-forming peptides had rarely beenexamined. Therefore, hamster prion peptide PrP(108-144) was used toexplore the association specificity of V24P(10-40). PrP(108-144) formedamyloid fibrils when incubated in 20 mM NaOAc buffer, pH 3.7/140 mMNaCl. Chen et al., Proc Natl Acad Sci USA (2002) 99:12633-8. As shown inFIG. 21, whether equimolar V24P(10-40) was added or not, thefluorescence intensity increase in the ThT binding assay showed thatPrP(108-144) formed amyloid fibrils during incubation, and V24P(10-40)failed to inhibit amyloid formation of PrP(108-144). These resultsindicated that the inhibitory effect of V24P(10-40) was target-specific.

Scavenger Peptide V24P(10-40) Decreased Aβ Accumulation in the Brains ofAPP/PS1 Transgenic Mice

In the design of peptide inhibitors, D-form amino acids, end capping,and methylation of amide hydrogens are often used to combat digestionsby exopeptidases and endopeptidases in the serum in order to extend thelifetime of the peptide in vivo. Findeis et al., Biochem (1999)38:6791-800; Gordon et al., Biochem (2001) 40:8237-45; Gordon et al., JPept Res (2002) 60:37-55. Modifications to putrescine, a naturallyoccurring polyamine, have been used to increase the ability of adesigned peptide D-YiAβ11 to cross the blood brain barrier. Poduslo etal., J Neurobiol (1999) 39:371-82. Polyethylenimine (PEI) cationizedproteins were also found to be able to cross the cell membrane. Futamiet al. J. Biosci. Bioeng. (2005) 99(2):95-103; Futami et al., Expert.Opin. Drug. Discov. (2007) 2:261-269; Kitazoe et al., J. Biochem. (2005)137:693-701; Kitazoe et al., Biotechnol. J. (2010) 5:385-392; Murata etal., J. Biochem. (2008) 144:447-455; Murata et al., J. Biosci. Bioeng.(2008) 105:34-38.

In this study, PEI was added to the C-terminus of the scavenger peptideV24P(10-40). 4-month-old APP/PS1 transgenic mice were administered withV24P(10-40)-PEI or PEI alone by nasal drops to both nostrils (1 nmole or4 μg per nostril) six times per week. The mice were then sacrificed 4months later and the A β contents of the brain were analyzed by ELISA.As shown in FIG. 22, the mice treated with V24P(10-40)-PEI clearly haddecreased A β40 and A β42 levels in both the cortex (panel A) andhippocampus (panel B), the effect being greater with Aβ40, probablybecause the scavenger peptide does not contain residues 41 and 42.

The effect of the scavenger peptide on reducing amyloid plaqueaccumulation was examined in APP/PS1 mice treated with V24P(10-40)-PEIor PEI from the age of 4 months to 12 months, using micro positronemission tomography (microPET) with the radiotracer ¹¹C-labeledPittsburg compound B ([¹¹C]PiB), which binds to A β plaques. ThemicroPET images (FIG. 23, panel A) taken at the end of treatment showedthat A β plaque deposition was decreased significantly by the peptidetreatment. Quantification of [¹¹C] PiB levels in the cortex,hippocampus, amygdala, and olfactory bulb in APP/PS1 mice with orwithout peptide treatment revealed that peptide treatment resulted in asignificant reduction in amyloid plaque formation in all four brainregions (FIG. 23, panel B).

Discussion

All the peptides with the V24→^(D)P replacement were less toxic thanAβ40 for N2a cells, suggesting that V24 is an important residue for Aβ40to form toxic species. In addition, the data showed that, without theC-terminal hydrophobic tail, V24P(1-28) still retained the ability toform amyloid fibrils as an Aβ 40 peptide, whereas peptides without theN-terminal hydrophilic tail increased the aggregation propensity ofV24P(10-40) to form amyloid-like β-aggregates. Shorter peptidesV24P(13-36), V24P(16-33), and V24P(19-30) had lower aggregationpropensity and a higher peptide concentration was required for them toform amyloid-like β-aggregates.

The results of these peptide inhibitors tested in the transgenic mousemodels described herein were summarized and compared in Table 4. In thepresent study, non-invasive intranasal administration and less peptide(˜200 μg of V24P(10-40)-PEI per mouse per month) were used. Treatmentfor four months resulted in a reduction of 72% and 40% in the Aβ40 andAβ42 content of the hippocampus, respectively. In terms of total peptideused in transgenic mouse models, 0.8 mg of V24P(10-40)-PEI was used,while previous studies used 2.5 mg of iAβ5p (icy infusion), 24 mg ofiAβ5p (ip), 0.27 mg of D3 (hippocampal infusion), 28-56 mg of D3 (oral),or 3 mg of D-Trp-Aib. The present peptide markedly reduced both the Aβlevel and amyloid accumulation in the brain at a much lower dosage whengiven via a non-invasive route. Thus, the present data shows thetherapeutic effects of the bipartite peptides described herein fordelaying the onset and/or reducing the progression of AD.

Questions have been raised about peptide therapy in terms ofimmunogenicity, low stability, low solubility, poor bioavailability, andlow BBB permeability. The present study proved that intranasaladministration is an effective method of delivering peptide drugs intothe brain. Since this administration route is much more user-friendlythan the invasive routes such as intracerebral infusion or ip injection,peptide drugs can be given daily in this manner. Intranasal peptideadministration could be used to deliver other peptide inhibitorstargeting other functions in the brain, for example, the 24-aa peptideTFPS derived from p35 (the cdk5 activator) which has been reported toreduce cdk5 hyperactivation and inhibit tau hyperphosphorylation in micevia ip injection. Shukla et al., FASEB J (2013) 27:74-86.

TABLE 4 Comparison of different peptides used in APP transgenic mice.Administration route and mouse age at Total Total Aβ in start and end ofpeptide peptide hippocampus Peptide treatment^(a) (mg) (μmol) (% ofcontrol) V24P (10-40)- Intranasal delivery 0.8 0.2 Aβ40 28%^(b) PEI 6times a week for 4 Aβ42 60% months; 4-m to 8-m V24P (10-40)- Intranasaldelivery 1.6 0.4 81%^(c) PEI 6 times a week for 8 months; 4-m to 12-miAβ5p (40) Icv infusion for 8 2.5 3.6 33%^(d) weeks; 6/7-m to 8/9-miAβ5p (40) Ip injection 24 34.6 54%^(d) 3 times a week for 8 weeks;8/9-m to 10/11-m D3 (42) Hippocampal 0.27 0.17 67%^(d) infusion for 30days; 8-m to 9-m D3 (43) Oral daily for 8 28-56 17.5-35 68%^(d) weeks;4-m to 6-m D-Trp- Ip injection 3 10.4 53%^(e) Aib (44) 3 times daily for120 days; 4.5-m to 8.5-m ^(a)intracerebroventricular (icv);intraperitoneal (ip) ^(b)quantified by ELISA ^(c)quantified by microPET^(d)quantified by immunohistochemistry ^(e)quantified by thioflavin Sstaining (40) Permanne et al. FASEB J. (2002) 16(8): 860-862. (42) vanGroen et al., ChemMedChem (2008) 3: 1848-1852. (43) Funke et al. ACSChem. Neurosci. (2010) 1(9): 639-648. (44) Frydman-Marom et al., Angew.Chem. Int. Ed. (2009) 48: 1981-1986.

(Relevant teachings of these references are incorporated herein byreference.)

MicroPET images showed that THE amyloid load in the olfactory bulb wassignificantly decreased. It has been reported that senile plaques andneurofibrillary tangles accumulate in the olfactory bulb and olfactionis damaged in the early stage of AD. Christen-Zaech et al., Can J NeurolSci (2003) 30:20-5; Arnold et al., Ann Neurol (2010) 67:462-9. In APPtransgenic mice, non-fibrillar Aβ deposition can be detected in theolfactory bulb earlier than in other brain regions, and olfactorydysfunction correlates with A β burden. Wesson et al., J Neurosci (2010)30:505-14. Thus, A β deposition in the olfactory epithelium may serve asa biomarker for identifying AD patients at the preclinical stage. Attemset al., Acta Neuropathol (2014) 127:459-75. Since the present peptidewas delivered to the brain intranasally, it has the advantage ofinhibiting Aβ deposition in the olfactory bulb at the early stage of ADprogression.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

1. A bipartite molecule comprising; (i) a peptide that binds an abnormalprotein aggregate associated with a disease or a component of theabnormal protein aggregate; and (ii) at least one charged moiety;wherein the peptide and the at least one charged moiety are conjugatedcovalently.
 2. The bipartite molecule of claim 1, wherein the abnormalprotein aggregate is associated with a neurodegenerative disease.
 3. Thebipartite molecule of claim 1, wherein the neurodegenerative disease isAlzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease(PD), Dementia with Lewy body (DLB), amyotrophic lateral sclerosis(ALS), Frontotemporal lobar degeneration-TDP-43 proteinopathy,Frontotemporal lobar degeneration-Tauopathy, Frontotemporal lobardegeneration with ubiquitinated inclusions (FTLD-U), Pick's disease,Cortical basal degeneration, Progressive supranuclear palsy, FTDP-17, orCreutzfeldt-Jacob disease (CJD).
 4. The bipartite molecule of claim 1,wherein the peptide comprises a fragment of amyloid β or TDP-43, whichinterferes with protein aggregation of amyloid β or TDP.
 5. Thebipartite molecule of claim 4, wherein the peptide comprises the aminoacid sequence GSNKGAIIGLM (SEQ ID NO: 1); orYEVHHQKLVFFAED^(D)PGSNKGAIIGLMVGGVV (SEQ ID NO: 2).
 6. The bipartitemolecule of claim 1, wherein the peptide is a polyglutamine (PolyQ)fragment.
 7. The bipartite molecule of claim 6, wherein the PolyQfragment consists of about 5-20 glutamine residues.
 8. The bipartitemolecule of claim 1, wherein the charged moiety is a polyarginine(PolyR) fragment or polyethylenimine (PEI).
 9. The bipartite molecule ofclaim 1, wherein the molecule comprises two charged moieties, one ofwhich is a polyR fragment and the other one is polyethylenimine (PEI).10. The bipartite molecule of claim 1, wherein the molecule is selectedfrom the group consisting of: (SEQ ID NO: 3) RRRRRRRRGSNKGAIIGLM,(SEQ ID NO: 4) RRRRRRRRGSNKGAIIGLM-PEI, (SEQ ID NO: 5)YEVHHQKLVFFAEDDPGSNKGAIIGLMVGGVV-PEI, (SEQ ID NO: 6)RRRRRRRRWDQQQQQQQQQQ,  and (SEQ ID NO: 7) RRRRRRRRWDQQQQQQQQQQQQQQQ.


11. A pharmaceutical composition, comprising a bipartite molecule ofclaim 1 and a pharmaceutically acceptable carrier. 12-15. (canceled) 16.A method for treating a disease associated with an abnormal proteinaggregate, the method comprising administering to a subject in needthereof an effective amount of the pharmaceutical composition of claim11.
 17. The method of claim 16, wherein the subject is a human patienthaving, suspected of having, or at risk for a neurodegenerative disease.18. The method of claim 17, wherein the neurodegenerative disease isselected from the group consisting of Alzheimer's disease (AD),Huntington's disease (HD), Parkinson's disease (PD), Dementia with Lewybody (DLB), amyotrophic lateral sclerosis (ALS), Frontotemporal lobardegeneration-TDP-43 proteinopathy, Frontotemporal lobardegeneration-Tauopathy, Frontotemporal lobar degeneration withubiquitinated inclusions (FTLD-U), Pick's disease, Cortical basaldegeneration, Progressive supranuclear palsy, FTDP-17, andCreutzfeldt-Jacob disease (CJD).
 19. The method of claim 18, wherein theneurodegenerative disease is Alzheimer's disease (AD) and the bipartitemolecule comprises a peptide that comprises the amino acid sequence ofGSNKGAIIGLM (SEQ ID NO: 1) or YEVHHQKLVFFAED^(D)PGSNKGAIIGLMVGGVV (SEQID NO: 2).
 20. The method of claim 16, wherein the bipartite moleculeis: (SEQ ID NO: 3) RRRRRRRRGSNKGAIIGLM, (SEQ ID NO: 4)RRRRRRRRGSNKGAIIGLM-PEI,  or (SEQ ID NO: 5)YEVHHQKLVFFAED^(D)PGSNKGAIIGLMVGGVV-PEI..


21. The method of claim 16, wherein the neurodegenerative disease isHuntington's disease and the bipartite molecule comprises a peptide thatcomprises a polyQ fragment.
 22. The method of claim 20, wherein thebipartite molecule is: (SEQ ID NO: 6) RRRRRRRRWDQQQQQQQQQQ,  or(SEQ ID NO: 7) RRRRRRRRWDQQQQQQQQQQQQQQQ.


23. The method of claim 16, wherein the bipartite molecule isadministered by an intranasal route.